Structure of the 30S translation initiation complex coupled to paused RNA polymerase and its potential for riboregulation

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Here, we present structural evidence from cryo-EM demonstrating that a bacterial RNA polymerase that is paused proximally to the promoter can associate with the pioneering 30S translation initiation complex (30S IC) through mutual binding of the transcription factor NusG. These findings suggest that the physical link between transcription and translation can be established prior to commitment to protein synthesis. Although the mRNA is embedded in this ‘early expressome’ complex, it can nonetheless interact with small regulatory RNA (sRNA) and be targeted for cleavage in the protein-coding region by the RNA degradosome assembly in vitro . The potential tagging of transcripts with sRNA during pioneering and subsequent stages of translation initiation, when the 30S IC is at the 5′ end of a polyribosome, may support surveillance processes that ensure efficient and rapid termination of gene expression in response to regulatory signals. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Molecular biology/RNA metabolism/RNA quality control ribosome RNA polymerase translation initiation complex nascent transcript co-transcriptional regulation gene expression transcription translation coupling sRNA mRNA surveillance expressome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In prokaryotic cells, the cytoplasmic milieu and chromatin intermingle. Consequently, the machinery of transcription and translation can interact and operate concurrently on the same nascent mRNA molecule (Blaha and Wade, 2022 , Webster and Weixlbaumer, 2021 , Woodgate and Zenkin, 2023 , Miller et al., 1970 ). Transcription-translation coupling (TTC) appears to occur extensively in representative gram-negative bacterial species, but its extent can be affected by growth conditions and varies in different bacterial lineages. For example, in some firmicutes there may be comparatively fewer encounters between RNA polymerase (RNAP) with the ribosome and TTC may be infrequent (Johnson et al., 2020 , Zhu et al., 2021 , Iyer et al., 2018 ). The physical and kinetic basis of TTC has been studied extensively in Escherichia coli , and structural data from cryo-EM has provided details of the component interactions in complexes formed between RNAP, the ribosomal 30S small subunit, and the fully assembled ribosomal 70S particle (Demo et al., 2017 , Kohler et al., 2017 , Wang et al., 2020 , Webster et al., 2020 , Fan et al., 2017 , Qureshi and Duss, 2025 ). Interactions between the elongating RNAP and 70S particles have been visualised in situ in Mycoplasma pneumoniae under conditions of antibiotic stress (O'Reilly et al., 2020 ), implicating the in vivo relevance of that transient assembly. Structural studies of the transcription-translation complexes, referred to as the ‘expressome’, reveal different configurations between RNAP and 70S are possible, e.g. in collided or coupled modes, that depend on the spacer length of the linking mRNA transcript and the recruitment of transcription factors NusG and NusA that can bridge the interface of the RNAP-70S to form an assembly (Webster et al., 2020 , Wang et al., 2020 , Kohler et al., 2017 , Qureshi and Duss, 2025 ). The occurrence of TTC likely depends on relative rates of transcription and translation, and the frequency of transcription pausing by the RNAP. After transcription initiation, mRNA transcript elongation is highly processive but discontinuous, with a strictly regulated balance between elongation and transcription pausing (Artsimovitch and Landick, 2000 ). The nascent mRNA itself can directly regulate sequence-dependent transcriptional pausing without requirement for any co-factors (Chan and Landick, 1993 , Dey et al., 2022 ). Transcript elongation of many genes is paused within the first 200 nts which serves early regulation mechanisms including the recruitment of the transcription factor NusG that facilitates TTC formation (Mooney et al., 2009 , Imashimizu et al., 2015 , Hatoum and Roberts, 2008 , Washburn et al., 2020 ). Promotor-proximal pausing could provide the window of opportunity required to complete translation initiation, which occurs on a timescale of (10–30) seconds (Shaham and Tuller, 2018 , Irastortza-Olaziregi and Amster-Choder, 2020 , Zhu et al., 2016 ). However, it remains unclear whether the establishment of TTC occurs only once the pioneering 70S ribosome catches up and collides with the paused RNAP, which may have detrimental effects on transcription fidelity (Wee et al., 2023 ). Recent structural evidence shows that RNAP can recruit the 30S ribosomal subunit through transcription factor NusG, potentially as an early step of translation initiation (Webster et al., 2024 ). Here, we have prepared Escherichia coli 30S translation initiation complexes (30S ICs), including initiation factors IF1, IF2 and IF3 and initiator formyl methionyl-tRNA, and solved cryo-electron microscopy (cryo-EM) structures of the 30S IC in two states. We further assembled 30S ICs on reconstituted transcription elongation complexes (TEC) with a long nascent mRNA that mimics a state of promoter-proximal transcription pausing. Cryo-EM single-particle analysis shows that NusG establishes a physical link between the 30S IC and the paused RNAP, despite the long (> 70 nts) mRNA spacer and at in vitro concentrations far below the proposed dissociation constant of the NusE-NusG interaction (Burmann et al., 2010 ). The physically coupled complex of paused transcription and translation initiation, or ‘early expressome’, may represent a transient precursor to mature expressomes in vivo . We explore if the early expressome can be the target for RNA-mediated regulation by testing if the 30S IC-recruited transcript can be accessed by small regulatory RNA (sRNA) that represses the expression of the target gene through base-pairing complementarity with the mRNA. We used the sRNA MicC that directs cleavage and turnover of the ompD transcript encoding the outer membrane protein OmpD in Salmonella Typhimurium. Assisted by the RNA chaperone Hfq, MicC recognizes ompD and induces downstream ompD -cleavage by the endoribonuclease RNase E (Pfeiffer et al., 2009 , Bandyra et al., 2012 ). Considering that ompD -targeting occurs in the coding region (codons 23–26) and that the ternary complex of MicC and Hfq on ompD does not prevent ribosome engagement or translocation (Pfeiffer et al., 2009 ), the 30S IC and a TEC paused immediately downstream of the recognition sequence for MicC could potentially be targets for riboregulation. We observe that the ompD transcript in the early expressome can be cleaved by the endoribonuclease RNase E within the multi-enzyme RNA degradosome at a defined site that is directed by the MicC sRNA. Although the rate of mRNA degradation is slower when the target transcript lies within the early expressome, the preference of RNase E for the sRNA-guided cleavage site over alternative, unspecific cleavage positions is increased, suggesting that the early expressome may help expose the seed pairing region in the mRNA for recognition by sRNA and Hfq. Our cryo-EM data indicates that the flexible tether between RNAP and 30S ribosomal subunit can allow the early expressome to accommodate transcript recognition and degradation close to the RNAP. Thus, based on evidence presented here and from other studies, we propose that the establishment of TTC may serve as a transient checkpoint for the nascent mRNA intermediate which, once tagged by sRNA, becomes the target for co-transcriptional degradation. Results Cryo-EM structure of E. coli 30S translation initiation complexes on the ompD mRNA We had previously prepared 30S IC on a 187 nts long fragment of the ompD transcript encompassing the 5'-UTR from position − 69 up to positions + 118 (with + 1 being A in the AUG start codon) and shown that the ompD -30S IC can be recognized by MicC-Hfq and targeted for deactivating cleavage at position + 83 by the RNA degradosome (Bandyra et al., 2024 ). Here, we again reconstituted 30S IC on ompD together with formyl methionyl-tRNA and initiation factors IF1, IF2 and IF3, and determined their structure by cryo-EM single particle analysis. The image processing resulted in two distinct structures of the ompD -30S IC that have either IF3 or initiator tRNA and IF2 recruited (Fig. 1 , Methods, Suppl. Figure 1, Table 1 ). Both structures contain well-defined density for the 30S ribosomal subunit, as well as IF1 and the mRNA which are recruited in previously described positions (Hussain et al., 2016 ). The mRNA is bound to the cavity that is formed between head and body regions of the 30S, and the Shine-Dalgarno sequence (SD) in the 5'-UTR forms a duplex with the anti-SD of the 16S rRNA. IF3 is bound at the 30S near IF1 and mRNA (Fig. 1 A,B) in an orientation corresponds with a previously reported model of the T. thermophilus 30S IC in ‘state 1’ (Hussain et al., 2016 ). This arrangement also agrees with a recent report of E. coli IF3-bound 30S in which the IF3-CTD is positioned at the P-site (Uday et al., 2023). Our second structure, which arose from the same specimen but was reconstructed from a separate particle subset, lacks IF3 but contains IF2 and the initiator tRNA in addition to IF1 and the 24 nts segment of the ompD mRNA (Fig. 1 C). The start codon of the mRNA forms triplet base pairs with the anticodon of the bound initiator tRNA that is bound in the P-site (Fig. 1 D), confirming that the 30S IC is located at the + 1 position of the ompD mRNA fragment. The arrangement of the IC components on the 30S ribosomal subunit is consistent with previously reported models of the T. thermophilus 30S IC in ‘state 4’ (Hussain et al., 2016 ). Additionally, our cryo-EM density resolves the N2 subdomain of the E. coli IF2 (residues 295–370) which attach to helix 16 of the 16S rRNA, resembling reported structures of P. aeruginosa IF2 at the 70S-IC (Basu et al., 2022) (Fig. 1 E). The head region of the 30S ribosomal subunit displays a lower degree of mobility in respect to the 30S body, compared to the structure that contains IF3 but lacks the initiator tRNA. Ribosomal S1 protein contacts the 5 ' -UTR at the shoulder of the 30S We observe additional cryo-EM density at the 5' mRNA exit site that allows unambiguous placement of the first two domains of S1 (residues 5-171) in an orientation that is in agreement with previous observations (D'Urso et al., 2023 ) (Fig. 1 C right, F ). Notably, the constellation of the S1 domains differs from available 70S ribosome and expressome structures (Webster et al., 2020 , Wang et al., 2020 , Loveland and Korostelev, 2018 ), but agrees with reports of the 30S-tRNA-RNAP complex (Webster et al., 2024 ), suggesting that S1 undergoes large conformational changes during translation initiation. At lower map-thresholds the density for S1 in our ompD -30S IC structure further extends from the described binding site along the shoulder of the 30S body towards proteins S6 and S11 and up to the binding site of IF3. The predicted model for the S1 protein in isolation (Varadi et al., 2022) shows an array of six OB fold-like S1-domains. The observed density in our 30S IC can accommodate additional S1-domains in an elongated arrangement and along with potentially bound 5'-UTR of the mRNA. This is consistent with reports of S1 interacting with upstream portions of mRNA on the 30S surface (Sengupta et al., 2001) and/or binding of A/U-rich segments that precede the Shine-Dalgarno sequence (Komarova et al., 2002 ). In a paused ompD transcription elongation complex, the seed pairing region remains accessible to sRNA. RNA polymerase can transiently pause during transcription. Genome-wide studies of in vivo transcriptional pausing in E. coli have identified a gene-encoded signal element with the consensus motif G − 10 Y −1 G + 1 (where − 1 corresponds to the 3'-end of the nascent RNA, with Y representing a pyrimidine base, Suppl. Figure 2A ) at which transcriptional pausing occurs with increased likelihood (Vvedenskaya et al., 2014 , Larson et al., 2014 , Imashimizu et al., 2015 ). With high conservation, the ompD gene harbours this sequence motif in its coding region, 28 nts downstream of the recognition sequence for the sRNA MicC ( Suppl. Figure 2B ). Two adjacent guanine bases at the upstream end (positions G − 9 and G − 10 within the motif) could stabilize the DNA-RNA hybrid of a paused TEC in pre- and post-translocated states (Imashimizu et al., 2015 ). In S. Typhimurium ompD , the G + 1 nucleotide of the pause motif lies at position + 106 in the coding region and is consistent with a putative paused transcript species of corresponding length that we observed when transcribing ompD in vitro using E. coli RNAP ( Suppl. Figure 2C-E ). To investigate whether transcription pausing at the described position impacts ompD recognition by Hfq/MicC and deactivating cleavage by the RNA degradosome, we reconstituted TECs in vitro with ompD segments encompassing the 5'-UTR (from position − 69) up to positions + 105 or + 99 ( Suppl. Figure 2F ). The 9 terminal nucleotides at the 3'-ends were altered to match the template DNA strand of a short, artificial transcription bubble for TEC reconstitution ( Suppl. Figure 2G ). We validated correct ompD -TEC assembly by cryo-EM (Fig. 2 A-C, Suppl. Figure 2H-L ). The 3D reconstruction shows clear density for two α, β and β’ subunits of the RNAP core enzyme, but none for the ω subunit (Fig. 2 B). As expected, the substrate binding site of the RNAP harbours the duplex DNA construct, which can be continuously traced through the cryo-EM density. The transcription bubble displays the melted base pairs of which 10 nucleotides in the template DNA are hybridised to the 3'-end of the mRNA (Fig. 2 C). The active site harbours an unpaired template nucleobase with RNAP in the post-translocated state. Though present in the sample and co-eluted from preparative size exclusion chromatography performed before sample vitrification, Hfq and MicC are not resolved in the cryo-EM map. Nevertheless, the structure shows that the ompD -TEC assembles as anticipated and demonstrates that the presence of MicC-Hfq does not disturb the complex. The reconstituted ompD 105 -TEC places the RNAP at the above-described putative pause element, 28 nts downstream of the MicC hybridisation site. Based on our structure and in silico docking with available molecular models of TEC (PDB: 2ppb) and RNase E (PDB: 2c0b) (Vassylyev et al., 2007 , Callaghan et al., 2005 ), a reconstituted ompD 105 -TEC would expose the cleavage site at position + 83 with six downstream nucleotides outside the RNAP exit channel and should thus be accessible to the active centre of RNase E. Conversely, a six nucleotides shorter ompD 99 -TEC should still allow the MicC seed region to hybridize to the recognition sequence in ompD , but the + 83-cleavage site would be masked by the RNAP exit channel and therefore sterically protected from RNase E cleavage. Indeed, ompD 99 -TECs were cleaved by RNase E at the MicC-induced + 83 cleavage site much less efficiently than free ompD 99 in the absence of RNAP ( Suppl. Figure 2M, N ), indicating correct TEC assembly at the 3'-end of the mRNA and the stability of the complex throughout exposure to regulatory RNA, Hfq and RNase E. The ompD 105 -TEC on the other hand remains accessible to MicC-induced cleavage at position + 83 by RNase E (Fig. 2 E) and the full RNA degradosome complex (Fig. 2 F). Moreover, cleavage at position + 83 appears more efficient when ompD 105 is a part of elongation complex, indicated by an increased rate of accumulation of the corresponding intermediate (Fig. 2 G). The + 72-cleavage product is an off-target species that is only observed in vitro (Pfeiffer et al., 2009 , Bandyra et al., 2012 , Bandyra et al., 2024 ). Cryo-EM structure of a coupled transcription elongation-translation initiation complex Promoter-proximal pausing of transcription in the coding region of genes after synthesis of the ribosome binding site could allow the translational and transcriptional machineries to synchronize. We explored whether the paused ompD -TEC could engage in physical coupling with the pioneering 30S ribosomal subunit during recruitment of translation initiation factors. As described above, we have successfully reconstituted TECs and 30S ICs separately and verified that the ompD -30S IC assembles at the start codon and demonstrated that the ompD -TEC assembles on the artificial transcription bubble at the 3'-end of the ompD -mRNA fragment, mimicking paused transcription elongation complexes. We then reconstituted both assemblies together on the ompD 105 transcript fragment in the presence of transcription factors NusA and NusG and performed cryo-EM analysis (Fig. 3 A, Suppl. Figure 3 ). Through iterative rounds of 3D classification, we selected 30S particles that showed clear signal for the initiation factors and initiator tRNA ( Suppl. Figure 3 ). The resulting map can accommodate the 30S IC, mRNA, initiator tRNA and IF1-3 in the positions that we observe in our 30S IC data, and we again observe the elongated density near the mRNA exit site that can be accounted for by the ribosomal S1 protein (Fig. 3 B). Additionally, our map displays a large area of unoccupied density on top of the 30S head which is not present in the ompD -30S IC structures. To improve the map in this area, we subtracted the signal for the 30S IC from the cryo-EM particles and performed iterative rounds of 3D refinement and classification, which enabled the isolation of a subset of particles that refined to fit the TEC portion of the coupled complex to ~ 6 Å resolution ( Suppl. Figure 3 ). The 30S-coupled TEC resembles the above-described structure of the ompD -TEC that was solved in the absence of the 30S IC but displays additional density for the ω subunit of RNAP as well as for the N-terminal domain (NTD) of transcription factor NusG. The NusG NTD is bound at the previously described binding site between b and b' subunits near the unwound non-template DNA strand (Kang et al., 2018 ) (Fig. 3 B). We also observe well-defined density for the NusG-CTD on the 30S ribosomal subunit near NusE (small ribosomal subunit protein S10) (Burmann et al., 2010 ), and this density is absent in our structures of the 30S IC that was prepared in the absence of NusG. Notably, density for the TEC appeared only in 3D-reconstructions generated from the subset of 30S particles that also contained signal for the initiation factors and initiator tRNA. Additionally, ompD 105 -TEC-IC samples prepared in the absence of NusG did not display physical coupling between TEC and 30S IC ( Suppl. Figure 4 ), suggesting that the physical coupling between RNAP and 30S IC is dependent both on NusG and mRNA-engagement by the small ribosomal subunit. When the cryo-EM particles of the final subset are aligned on either 30S IC or RNAP, the respective other component remains only visible as noisy density (Fig. 3 C, D; Suppl. Figure 3 ). This high mobility between TEC and 30S IC within the early expressome likely results from the long mRNA linker in which the start codon at the P-site of the 30S IC and the 3'end at the TEC lie almost 90 nucleotides apart. Nevertheless, the general orientation between TEC and 30S IC is consistent with the ‘coupled’ state that was described for expressomes with mRNA linkers of 8–10 codons, while complexes with shorter mRNA linkers position the TEC closer to the 70S mRNA entry site in a ‘collided’ complex that is incompatible with NusG recruitment (Wang et al., 2020 , Webster et al., 2020 , Kohler et al., 2017 ). Overall, our structural data demonstrates that a putative early expressome can be formed in vitro from a TEC and a 30S IC, and the physical coupling of the two subcomplexes relies on NusG which forms a link between mRNA-engaged 30S and RNAP. The coupled transcription elongation-translation initiation complex can be accessed at a target site by sRNA for effector recruitment The linker mRNA in the early expressome model loops out between the TEC and 30S IC, which may provide an opportunity for sRNA pairing to a target site. To test whether the ompD -mRNA, when embedded in the TEC-IC, is still accessible for recognition by MicC and deactivating cleavage by the RNA degradosome, we performed degradation reactions with the RNA degradosome in the presence of 5'P-MicC and Hfq (Fig. 4 , Suppl. Figure 5) . Alongside, we also subjected the free ompD 105 segment as well as the individual sub-assemblies ompD 105 -TEC and ompD 105 -30S IC to degradation reactions. The degradation rates of ompD 105 decreased with rising complexity of the assembly, down to a 4-fold slower degradation of NusG-containing ompD -TEC-IC compared to free ompD (Fig. 4 B, C). Double-exponential equation fitting of the formation of the + 83-cleavage intermediate and degradation of ompD 105 is shown in Fig. 4 B, C. The formation rate allows comparison of cleavage specificity for the + 83 site which is expressed as the formation rate of the + 83 intermediate over the degradation rate of the ompD 105 starting material. Cleavage specificities for the + 83 site in the different ompD 105 assemblies are shown in Fig. 4 D. The RNA degradosome shows the lowest preference for + 83-cleavage on free ompD 105 and the highest on the ompD 105 -TEC-IC substrate. The fraction of the unspecific + 72-cleavage intermediates on the other hand is highest in the case of free ompD 105 , reduced for the ompD 105 -TEC and -IC and was unquantifiable in the early expressome ( Suppl. Figure 5 ). The observation that MicC-guided processing by RNase E occurs with higher specificity when RNAP and/or 30S IC are engaged with the ompD mRNA suggests that the gene expression machineries might facilitate the recognition of ompD by the sRNA and/or present the MicC- ompD hybrid in a way that increases susceptibility to attacks by the RNA degradosome at the + 83cleavage site. The cryo-EM maps of the ompD 105 -TEC-IC complex reveal conformational sub-states that may facilitate regulatory access of the sRNA to the seed pairing site in the transcript (Fig. 4 E, F). One of the modes of conformational variation is in a relative tilting of the RNAP with respect the 30S IC, and this is correlated with additional density on the exposed segment of the mRNA ( right panel , Fig. 4 E ) . The density is not sufficiently resolved to build a detailed model, but the shape of the envelope can accommodate Hfq and may represent a transient species in which the sRNA/Hfq interacts with the exposed seed region. The schematic in Fig. 4 F illustrates a potential model for this transient access. Discussion In this study, we have explored the accessibility of a small RNA pairing site in a target transcript at early stages of transcription elongation and translation initiation. We reconstituted transcription elongation complexes (TEC) mimicking a state of promoter-proximal transcription pausing of the target mRNA and assembled the 30S translation initiation complex (30S IC) on those mRNAs. Cryo-EM single-particle analysis shows that the transcription factor NusG can physically link the 30S IC and the paused TEC, despite the lengthy mRNA linker (> 70 nts) and the in vitro concentrations used that are far below the proposed dissociation constant of the NusE-NusG interaction (Burmann et al., 2010 ). The physically coupled assembly, which we refer to as an ‘early expressome’, may represent a transient precursor to mature expressomes in vivo . During the preparation of this manuscript, an independent study reported the in vitro reconstitution of similar transcription elongation-translation pre-initiation complexes (Webster et al., 2024 ). Webster et al. ( 2024 ) present structures of mRNA delivery complexes between a paused TEC and 30S ribosomal subunit in the inactive states and propose two pathways that initiate the coupling between transcription and translation through transcription-assisted recruitment of mRNA to the ribosome (Webster et al., 2024 ). One pathway involves ribosomal protein S1 of the 30S particle interacting with the TEC and binding the nascent mRNA to form an intermediate or standby complex that directs the nascent transcript to the Shine-Dalgarno interaction site of the 30S. The RNAP is transiently located near the cluster of S1 OB-domains and mRNA exit site of the 30S and subsequently repositioned to be near the mRNA entry site in the translational 30S IC. The second pathway involves RNAP directly binding near the mRNA entry site of the inactive 30S, which then transitions to the active form in the assembled 30S IC by folding the 16S rRNA helix 44 away from the mRNA exit channel into its active position on the 30S body (Webster et al., 2024 ). Our data include IF1-3 and present the 30S IC in ‘accommodated’ active states which can be formed via both proposed pathways. The structural data presented here also corroborate how the ribosomal S1 might be poised to facilitate interactions with 5'-UTR elements. Webster et al. ( 2024 ) report that the S1 protein not only facilitates mRNA delivery to selectively accelerate duplex formation with the anti-Shine-Dalgarno sequence but helps RNAP to stimulate translation initiation (Webster et al., 2024 ). S1 assists the remodelling of mRNA secondary structures in vitro to aid the 30S with mRNA loading and 30S IC formation (Duval et al., 2013 , Kolb et al., 1977 ). When the ompD mRNA is recruited to the 30S IC, RNase E relies on the assembly of the RNA degradosome for access and efficient MicC-guided degradation of the ompD transcript (Bandyra et al., 2024 ). Ribosomal S1 has been shown to form direct interaction with several sRNAs from E. coli (Windbichler et al., 2008 ), thereby possibly exerting the 30S IC’s influence on Hfq-MicC-guided RNase E attack while located on the 30S ribosome. Further, direct interactions with the TEC (Sukhodolets and Garges, 2003 ) might also facilitate riboregulation at the early stage of nascent transcript recruitment to the translation machinery. The paused RNAP may increase the recognition efficiency of the target element within ompD by Hfq-MicC, consistent with our observation that ompD -cleavage by RNase E is more site-specific at the paused TEC. This is supported by a recent single-molecule study showing that co-transcriptional target recognition by sRNA and Hfq close to the RNAP exit channel is more efficient than recognition post transcription (Rodgers et al., 2023 ). The biogenesis of 3'-UTR-derived sRNAs that autoregulate their genes (Hoyos et al., 2020 ), along with kinetic modelling studies, provide further support for mechanisms of co-transcriptional regulation by sRNAs (Reyer et al., 2021 ). Access to decay machinery during transcription is also supported by observations of co-transcriptional stabilisation of riboswitches against ribonuclease action (Lou and Woodson, 2024 ). Moreover, a recent study presents a model of co-transcriptional endonucleolytic cleavage of RNA in archaea that leads to transcription termination (You et al., 2024 ), suggesting the possibility of a functionally analogous system in that domain of life. In the early expressome of the ompD transcript in which the RNAP is paused at position + 106, the deactivating cleavage site at position + 83 for RNase E that is preceded by the recognition site for the MicC sRNA, remains accessible to regulatory RNA and deactivating cleavage by the RNA degradosome in in vitro reactions. The cellular location of the degradosome must be considered to explain how access might be gained to the putative TEC-30S IC complex tagged with a small RNA (Fig. 5 ). The envisaged recognition event, in which sRNA recruits degradosome to the 30S IC or TEC-IC complex, could occur in species such as Caulobacter crescentus , in which the RNA degradative machinery can be associated with the nucleoid or ribonucleoprotein condensates in the cell interior and could have access to nascent mRNA targets (Kim et al., 2024 ) (Fig. 5 i). However, in species such as Escherichia coli and Salmonella where the degradosome is compartmentalized to the cytoplasmic membrane, and potentially distant from the nucleoid (Carpousis et al., 2022 ), the sRNA-tagged early expressome might be the target for other effectors, such as Rho RNA translocase that terminates transcription through allosteric manipulation of the elongation complex (Molodtsov et al., 2023 , Said et al., 2021 , Hao et al., 2021 , Reyer et al., 2021 ). Rho can act to trigger premature transcription termination in the absence of transcription-translation coupling (Wang and Artsimovitch, 2020 , Kim et al., 2024 ) and its activity on nascent mRNA can be modulated by sRNA (Sedlyarova et al., 2016 ). Transcription-terminated transcripts could then encounter the degradosome on the membrane upon their diffusion following the release from RNAP (Fig. 5 i i ). Another scenario in which membrane-bound RNase E can act early on nascent transcripts is if the transcriptional-translational machinery is brought close to the membrane, as occurs in the ‘transertion’ mechanism which was described for Vibrio parahaemolyticus and suggested to occur also in E. coli (Woldringh, 2002 , Bakshi et al., 2012 , Fishov and Norris, 2012 , Bakshi et al., 2014 , Roggiani and Goulian, 2015 , Kaval et al., 2023 ). Transertion, a coupling of transcription, translation and membrane insertion at the membrane component of the type III secretion system, is a process suggested to be common for bacterial membrane proteins. Evidence indicates that membrane-associated RNase E can act on transcripts encoding membrane proteins in E. coli to result in co-transcriptional degradation (Fig. 5 iii ) (Kim et al., 2024 ). A fourth scenario in which we envisage RNase E to act on a mRNA-30S IC complex is if the leading edge of a polyribosome comes into proximity of the membrane (Fig. 5 iv ). Earlier studies have shown that RNase E and the degradosome can interact with polyribosomes in vitro and potentially in vivo , and it was proposed that this forms a passive complex that does not cleave the RNA until activated by a signal, such as a cognate sRNA (Tsai et al., 2012 ). In species without RNase E, similar processes are likely to occur, such as in the model firmicute species Bacillus subtilis , where the membrane-bound degradosome is based on the distinct ribonuclease RNase Y (Laalami et al., 2024 ). This mode of degradation would enable the co-translational decay of a transcript. It also represents a more economical means of exiting translation, because it avoids generating transcript fragments that lack stop codons and entail the hidden metabolic costs of using the tmRNA system for rescue recovery. The above mechanisms could account for processes of co-translational decay (Huch et al., 2023 , Herzel et al., 2022 ) analogous to the recruitment of the 5'-to-3' exoribonuclease Xrn1 which follows the terminal translating ribosome identified in yeast and other eukaryotic species (Pelechano et al., 2015 , Tesina et al., 2019 ). Material and Methods Protein production Wild-type 30S subunits were prepared from E. coli MRE600 strain using zonal centrifugation (Rodnina et al., 1994, Rodnina and Wintermeyer, 1995 ). Initiator tRNA (fMet-tRNAfMet) and E. coli initiation factors IF1, IF2, and IF3 were purified according to published protocols (Milon et al., 2007 ). E. coli Hfq was purified as described (Dendooven et al., 2021 ). The RNA degradosome was prepared as described (Dendooven et al., 2022 ). E. coli core RNAP was expressed from pVS10 plasmid coding for all five subunits (Svetlov and Artsimovitch, 2015 ) and purified as described (Castro-Roa and Zenkin, 2015 ). RNA Production RNAs were prepared by in vitro transcription (IVT) as described (Bandyra et al., 2012 ). In brief, IVT templates were amplified by PCR from plasmid pVP042-3 that carries the Salmonella Typhimurium ompD gene (Pfeiffer et al., 2009 ). The forward primer introduces a T7 promoter sequence upstream of the transcription initiation site and, where indicated, the reverse primer encodes a non-canonical 9 nts sequence (CGGCGCUGG) at the 3’ end of the corresponding transcript. IVT reactions containing 3 µg of template DNA, 5 mM of each of ATP, UTP, GTP and CTP, 10 mM DTT and 0.5 U/µL RNaseOUT™ (Invitrogen) were incubated with recombinant T7 RNA polymerase in 40 mM Tris, pH 8.0, 25 mM MgCl 2 , 2 mM spermidine at 37°C for 5 h. IVT products were DNase I-treated, resolved by 4% denaturing PAGE and excised RNA bands were electroeluted using Elutrap™ Electroelution System Kit (Whatman), followed by clean-up using PureLink™ RNA Micro Kit (Invitrogen). Assembly of transcription elongation complexes TECs were assembled following a previously described strategy (Said et al., 2021 ). To anneal nucleic acid scaffolds, 8 µM mRNA fragment was mixed with template DNA (tDNA) in a 1:1.25 molar ratio in 10 mM TRIS, pH 7.6, 40 mM KCl, 5 mM MgCl 2 , heated to 95°C for 2 min and slowly equilibrated to 37°C. Two volumes TEC reconstitution buffer (20 mM TRIS, pH 7.6, 120 mM potassium acetate, 5 mM magnesium acetate, 1 mM TCEP) were pre-heated to 37°C and added to annealed RNA/tDNA. E. coli RNAP was added in 1:1 ratio to RNA, followed by 10 min incubation. Non-template DNA was added in 1:1 ratio to tDNA and followed by incubation for 10 min at 37°C. Assembled TECs were prepared fresh and used immediately for degradation assays or assembly of higher order complexes. For cryo-EM specimen preparation, 5 µM TEC was mixed with equimolar amount of pre-incubated (32°C for 30 min) MicC and Hfq injected onto a 3.2/300 Superose 6 size-exclusion chromatography column equilibrated with 20 mM TRIS-Cl, pH 7.6, 120 mM potassium acetate, 5 mM magnesium acetate, 2 mM DTT, 10 µM ZnCl 2 . Fractions containing all components as determined by urea-PAGE and SDS-PAGE were pooled, concentrated to 40 µL and used directly for vitrification. Preparation of ompD -30S IC and early expressomes 30S IC was prepared following a previously described strategy (Goyal et al., 2017 ). In brief, 30S subunits were incubated in buffer TAKM 20 (50 mM Tris-HCl [pH 7.5], 70 mM NH 4 Cl, 30 mM KCl, 20 mM MgCl 2 ) for 30 min at 37°C for reactivation. Reactivated 30S subunits were incubated with a 2.5-fold excess of mRNA, 2-fold excess of initiation factors 1–3 and a 2.5-fold excess of initiator fMet-tRNA fMet (hereafter tRNA) in the presence of 250 µM GTP or GTPγS (Jena Bioscience) in TAKM 7 buffer (Goyal et al., 2017 ). For transcription/translation-coupled complexes, the 30S IC was prepared as described above using pre-assembled ompD -TEC instead of free mRNA and omitting the chromatography step. Coupled TEC-IC for cryo-EM analysis was prepared with final concentrations 0.3 µM 30S, 1.2 µM TEC, 0.6 µM of IFs and tRNA, 10 µM NusA, 20 µM NusG and 5 µM MicC/Hfq and directly used for grid preparation. In vitro transcription The first 306 bases of the ompD gene (-69 to + 237) were inserted into the pMMB67HE vector between tac promoter and T1 terminator (Furste et al., 1986 ). For in vitro transcription assays, 15 µL reactions containing 5 nM ompD -pMMB67HE, 0.5 U/µL RNaseOUT™ and 0.5 U E. coli RNA Polymerase Holoenzyme (NEB, M0551S) were pre-incubated in reaction buffer (40 mM Tris-HCl, pH 7.5, 150 mM KCl, 10 mM MgCl 2 , 1 mM DTT, 0.01% Triton X-100™) at 37°C for 5 minutes. Reactions were started by adding ribonucleotide mix to 5 mM, incubated at 37°C for 1 h and quenched by addition of equal volume stop buffer (200 mM Tris-HCl, pH 7.5, 25 mM EDTA, 300 mM NaCl, 2% SDS, 0.5 mg/mL Proteinase K). After proteolysis at 50°C for 1 h, samples were mixed with 0.5 volumes 2x RNA Loading Dye (Thermo Scientific), heated to 95°C for 2 min and separated by urea PAGE on 8% polyacrylamide gels. Graphene oxide coating and grid preparation Graphene oxide (GO) coating was prepared by an adaptation of the drop-casting method (Pantelic et al., 2010 ). Quantifoil Cu 300 1.2/1.3 grids were glow-discharged on the darker carbon side (PELCO easiGLOW: 15 mA, 0.28 mBar, 2 min). GO solution (Sigma-Aldrich 763705, 2 mg/mL dispersion) was diluted 10-fold in water and centrifuged at 300 xg for 30 s to pellet insoluble GO flakes. The supernatant was further diluted 10-fold to a working concentration of 0.02 mg/mL. 1 µL working solution was applied to the glow-discharged side of the Quantifoil grids and dried at room temperature for 10 min. GO-coated grids were kept at room temperature for at least 16 h and then directly used for sample vitrification. Quantifoil 1.2/1.3 grids with a 2 nm amorphous carbon support layer were briefly glow-discharged (PELCO easiGLOW: 10 s, 25 mA, 0.39 mBar) directly before sample application. For vitrification, 4 µL of sample was applied to grids and incubated for 30 s. Using a FEI Vitrobot, excess sample was blotted, and grids were plunge-frozen in liquid ethane. Grids were subsequently stored in liquid nitrogen until screening and collection. Cryo-EM data collection and processing For structures of ompD -30S IC, 9104 multi-frame movies were collected on a 300 kV FEI Titan Krios equipped with a Gatan K3 detector. For the TEC structure, a dataset of 1619 multi-frame movies was collected on a 300 kV Titan Krios equipped with a Falcon 3 detector in Counting mode. For the TEC-30S IC structure, 12011 multi-frame movies were collected on a 300 kV FEI Titan Krios equipped with a Gatan K3 detector. Details for data collection are summarised in Supplementary Table 1 . Mult-frame movies were motion-corrected in Relion 3.1 (Scheres, 2012 ) and the generated micrographs were imported in cryoSPARC v4 (Punjani et al., 2017 ) and further processed as illustrated in Suppl. Figures 1–3 . For structures of TEC-30S IC assemblies, small datasets were collected on a 200 kV Talos Arctica equipped with a Falcon 3 detector in Counting mode and processed in Relion 3.1 (Scheres, 2012 ) as depicted in image processing workflows ( Suppl. Figure 4B, G, L ). Declarations Data Availability Data supporting the findings of this study are available from the PDB and EMDB. Acknowledgements We thank Andrzej Szewczak-Harris and the staff of the Cryo-EM Facility, Dimitri Y. Chirgadze, Steven Hardwick and Lee Cooper, for assistance with data collection. We thank the staff at eBIC for access to facilities and help with data collection. We thank Tom Dendooven, Kathi Frohlich, and Joerg Vogel for helpful comments and suggestions. 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Real time determination of bacterial in vivo ribosome translation elongation speed based on LacZalpha complementation system. Nucleic Acids Res, 44 , e155. ZHU, M., MU, H., HAN, F., WANG, Q. & DAI, X. 2021. Quantitative analysis of asynchronous transcription-translation and transcription processivity in Bacillus subtilis under various growth conditions. iScience, 24 , 103333. Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 13 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5868712","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":413324849,"identity":"43ffaaa3-4d40-4a38-90fe-c1a91b027801","order_by":0,"name":"Ben Luisi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYBACA+bDjQ8SKuD8BCK0sCU2G3w4Q6KWNsmZbaRoMWdjbJDmnWcnr9vA/PADY1saYS2WbYwNxrzbkg23HWAzlmBsyyHCYfcbG5J5tx1g3HaAwYyBsa2CCC3HGBsO8845YL/tAPs3orU0Ns5sOJC47QAPyBYiHAb0SzPDh2PJydsO8xRLJJwjwvvmbMzHfyTU2NluO96+8cOHsmTCWhCAmYGoWBkFo2AUjIJRQAwAANgKOdx+URftAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1144-9877","institution":"University of Cambridge","correspondingAuthor":true,"prefix":"","firstName":"Ben","middleName":"","lastName":"Luisi","suffix":""},{"id":413324850,"identity":"5fee3f68-275f-4572-9232-00aa52867313","order_by":1,"name":"Johann Roske","email":"","orcid":"https://orcid.org/0000-0001-8500-901X","institution":"MRC Laboratory of Molecular Biology","correspondingAuthor":false,"prefix":"","firstName":"Johann","middleName":"","lastName":"Roske","suffix":""},{"id":413324851,"identity":"e49c1bc6-dd4d-4334-9712-93bb816b4450","order_by":2,"name":"Giulia Paris","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Paris","suffix":""},{"id":413324852,"identity":"c6f858af-a94a-4f12-8e97-5d57820c591d","order_by":3,"name":"Akanksha Goyal","email":"","orcid":"","institution":"Max Planck Institute for Biophysical Chemistry","correspondingAuthor":false,"prefix":"","firstName":"Akanksha","middleName":"","lastName":"Goyal","suffix":""},{"id":413324853,"identity":"49946301-253e-4674-8392-7004a4f316d0","order_by":4,"name":"Marina Rodnina","email":"","orcid":"https://orcid.org/0000-0003-0105-3879","institution":"MPI-NAT","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"","lastName":"Rodnina","suffix":""},{"id":413324854,"identity":"a24206bd-48fa-441d-a08a-35e97112264e","order_by":5,"name":"Nikolay Zenkin","email":"","orcid":"https://orcid.org/0000-0003-2212-9545","institution":"Newcastle University","correspondingAuthor":false,"prefix":"","firstName":"Nikolay","middleName":"","lastName":"Zenkin","suffix":""},{"id":413324855,"identity":"69bcc113-5d9b-43a3-8b23-95ea806d1947","order_by":6,"name":"Katarzyna Bandyra","email":"","orcid":"","institution":"University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Bandyra","suffix":""}],"badges":[],"createdAt":"2025-01-20 21:35:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5868712/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5868712/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67330-2","type":"published","date":"2025-12-13T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":76005495,"identity":"c94f9d5f-8e19-480b-9e6a-fcc0227a2e0a","added_by":"auto","created_at":"2025-02-11 11:17:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1585052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e translation initiation complex (30S IC) on the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eompD\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transcript.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cryo-EM structure of the 30S IC on the \u003cem\u003eompD\u003c/em\u003e-mRNA with IF1 and IF3.\u003c/p\u003e\n\u003cp\u003e(B) Isolated view of the cryo-EM densities around the \u003cem\u003eompD\u003c/em\u003e mRNA and IF1/IF3 which are shown as ribbon models.\u003c/p\u003e\n\u003cp\u003e(C) Views of the cryo-EM structure of the 30S IC on the \u003cem\u003eompD\u003c/em\u003e-mRNA with IF1, IF2, and initiator tRNA (tRNA\u003csup\u003efMet\u003c/sup\u003e). The rotation symbol indicates the view of the left panel in respect to panel A.\u003c/p\u003e\n\u003cp\u003e(D) Close view of the cryo-EM density of the codon-anticodon pair between the \u003cem\u003eompD\u003c/em\u003e mRNA and the initiator tRNA.\u003c/p\u003e\n\u003cp\u003e(E) Isolated view of the cryo-EM density for IF1 and IF2. Inset: Molecular model for the N2 subdomain of IF2 with the contacting region of the 16S rRNA near ribosomal protein S4.\u003c/p\u003e\n\u003cp\u003e(F) Molecular model of the first two domains of the ribosomal S1 protein. The cryo-EM density is shown as a mesh around S1.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5868712/v1/6265dbbe335611905cdb2661.png"},{"id":76007136,"identity":"450c9902-59b7-4c05-a174-a60024b1439c","added_by":"auto","created_at":"2025-02-11 11:33:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1095525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe paused \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eompD\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-transcription elongation complex (TEC) can be directed by small regulatory RNA for cleavage by RNase E.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Cryo-EM structure of the reconstituted \u003cem\u003eompD\u003c/em\u003e-TEC. (A) Representative 2D-class averages generated from the final particle set, the scale bar indicates 10 nm. (B) 3D-refined and locally filtered cryo-EM map of the \u003cem\u003eompD\u003c/em\u003e-TEC structure. Subunits of RNAP and bound DNA and mRNA are indicated. (C) Isolated view on the RNAP active site. The unwound template DNA strand is shown in red, the nascent mRNA in green. The bridge helix feature of the RNAP b’-subunit at the active site is depicted in ribbon representation. The rotation symbols indicate the view in respect to panel B.\u003c/p\u003e\n\u003cp\u003e(D) 3' sequence of the \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e mRNA construct. The 3'‑terminal nucleotides that hybridize with the template DNA strand within the TEC are shown in bold black letters. A green oval delineates the 3'-terminal portion of the mRNA that is masked by RNAP in the reconstituted TEC. The recognition sequence for MicC is marked in purple. RNase E cleavage sites observed in \u003cem\u003ein vitro\u003c/em\u003e degradation reactions are annotated at positions +72, +83.\u003c/p\u003e\n\u003cp\u003e(E-G) Time course reactions of \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-mRNA or \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC incubated with MicC/Hfq and either RNase E (E) or the full RNA degradosome (F). Cleavage intermediates of \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e \u003c/em\u003eand MicC are indicated. (G) The relative abundances of the transient \u003cem\u003eompD\u003c/em\u003e cleavage intermediates from reactions shown in (F) were quantified (mean ± SD from three independent reactions) and plotted over time.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5868712/v1/56cad5c4d1968d58b940d3f8.png"},{"id":76005505,"identity":"74a80fe9-acfb-455f-8737-fd0a978c9aa3","added_by":"auto","created_at":"2025-02-11 11:17:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1712293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe structure of the transcription elongation-translation initiation complex with regulatory element in the bridging mRNA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of the reconstitution procedure for early expressomes \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(B) Composite cryo-EM map (top) and molecular model (bottom) of the ‘early expressome’, comprising NusG-coupled transcription elongation (\u003cem\u003eompD\u003c/em\u003e-TEC) translation initiation (and \u003cem\u003eompD\u003c/em\u003e-30S IC) complexes. Complex components that correspond to segments of the map are indicated. The lower panel shows molecular model interpretation of the cryo-EM structure based on reported structures for the translation initiation complex (PDB: 5lmv) and the NusG-coupled expressome (PDB: 6xgf) (Hussain et al., 2016, Wang et al., 2020).\u003c/p\u003e\n\u003cp\u003e(C, D) 2D class averages of cryo-EM particles that were aligned on either 30S or RNAP components of the early expressome assembly.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5868712/v1/64ec7892c2666d9f00a0a443.png"},{"id":76006699,"identity":"8dcc6f86-6f6b-4576-8f55-e72e8f830a66","added_by":"auto","created_at":"2025-02-11 11:25:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":844996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe bridging element in the TEC-IC complex is accessible to the RNA degradosome.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of the \u003cem\u003eompD\u003c/em\u003e transcript and MicC recognition site.\u003c/p\u003e\n\u003cp\u003e(B, C) Graphs displaying relative abundances of \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e mRNA and +83-cleavage intermediate species from time-course experiments (see \u003cstrong\u003eFigure 2F\u003c/strong\u003e and \u003cstrong\u003eSuppl. Figure 5\u003c/strong\u003e). Free \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e mRNA (black), \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC (green), \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-30S IC (grey) and \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC-IC (red) were incubated with MicC/Hfq and then subjected to cleavage by the RNA degradosome. Reactions were stopped at indicated time points and analysed by denaturing PAGE. Data are mean ± SD from three independent reactions. Degradation trajectories of \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e were fitted to one phase exponential decay. Formation and decay of intermediates from \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-cleavage at position +83 were fitted to two-exponential equation [I=exp(‑\u003cem\u003ekt\u003c/em\u003e) – exp(‑\u003cem\u003ejt\u003c/em\u003e)] with degradation rate \u003cem\u003ek\u003c/em\u003e and formation rate \u003cem\u003ej\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(D) Cleavage specificities for nucleolytic processing of site +83 in \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e in the different assembly states, expressed as the formation rate of the +83 intermediate over the degradation rate of the \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e starting material. Data are mean ± SD from three independent reactions.\u003c/p\u003e\n\u003cp\u003e(E) Conformational substates of the TEC-30S IC assembly.\u003c/p\u003e\n\u003cp\u003e(F) Substate of the early expressome proposed to facilitate the accommodation of Hfq/MicC on the bound \u003cem\u003eompD\u003c/em\u003e mRNA. The colours of the segmented map in E correspond to the components shown in the schematic.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5868712/v1/542ff4bd1e521c8f87f962a1.png"},{"id":76006698,"identity":"9e8e2cf7-f18c-427e-8a69-a2454c4ea5b3","added_by":"auto","created_at":"2025-02-11 11:25:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":412333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic models for sRNA mediated degradation of mRNAs during transcription and translation. \u003c/strong\u003eThe physical localization of the RNA degradosome to the membrane in \u003cem\u003eEscherichia coli\u003c/em\u003e and related species poses a spatial layer to the regulation of gene expression and the hypothesis of RNA surveillance. In this cartoon, four modes are presented where the RNA degradosome could access transcripts tagged with sRNAs and engaged with ribosomes and translating polysomes. Mode (i), proposed in this study, posits access to the pioneering 30S translation initiation complex in the vicinity of the paused polymerase elongation complex. This mode could occur in species such as \u003cem\u003eCaulobacter crescentus\u003c/em\u003e and \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e. In mode (ii) the RNA degradosome could act to turnover transcripts that might be incomplete through transcription termination. (iii) Genes encoding for some membrane proteins are known to be transcribed and translated in proximity to the membrane, following a process called transertion (Kaval \u003cem\u003eet al.\u003c/em\u003e 2023). When transertion occurs, the RNA degradosome bound to the inner membrane is close to the translation site and can interact with polysomes scanning for unbound mRNA to cleave. In mode (iv), the degradosome could be interacting with polysomes and once the mRNA has been translated, upon binding of Hfq:sRNA complexes, it could cleave the mRNA. This mode is supported by the \u003cem\u003ein vivo\u003c/em\u003e observation of RNA cluster formation by degradosomes in the presence of polysomes (Hamouche \u003cem\u003eet al.\u003c/em\u003e2021).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5868712/v1/5dff5303aceb4033aaf87565.png"},{"id":100765771,"identity":"2c69b983-e624-4f33-9ebf-c43f2db419b8","added_by":"auto","created_at":"2026-01-21 08:55:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7852379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5868712/v1/eee427ee-6ce7-4566-b808-235004c6d09c.pdf"},{"id":76006702,"identity":"fd54bd2e-897f-4d61-80f1-6891faad3eb0","added_by":"auto","created_at":"2025-02-11 11:25:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6485019,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5868712/v1/d751b6730b79ece622a4ab46.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Structure of the 30S translation initiation complex coupled to paused RNA polymerase and its potential for riboregulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn prokaryotic cells, the cytoplasmic milieu and chromatin intermingle. Consequently, the machinery of transcription and translation can interact and operate concurrently on the same nascent mRNA molecule (Blaha and Wade, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Webster and Weixlbaumer, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Woodgate and Zenkin, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Miller et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). Transcription-translation coupling (TTC) appears to occur extensively in representative gram-negative bacterial species, but its extent can be affected by growth conditions and varies in different bacterial lineages. For example, in some firmicutes there may be comparatively fewer encounters between RNA polymerase (RNAP) with the ribosome and TTC may be infrequent (Johnson et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zhu et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Iyer et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The physical and kinetic basis of TTC has been studied extensively in \u003cem\u003eEscherichia coli\u003c/em\u003e, and structural data from cryo-EM has provided details of the component interactions in complexes formed between RNAP, the ribosomal 30S small subunit, and the fully assembled ribosomal 70S particle (Demo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Kohler et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Wang et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Webster et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Fan et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Qureshi and Duss, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Interactions between the elongating RNAP and 70S particles have been visualised \u003cem\u003ein situ\u003c/em\u003e in \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e under conditions of antibiotic stress (O'Reilly et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), implicating the \u003cem\u003ein vivo\u003c/em\u003e relevance of that transient assembly. Structural studies of the transcription-translation complexes, referred to as the \u0026lsquo;expressome\u0026rsquo;, reveal different configurations between RNAP and 70S are possible, \u003cem\u003ee.g.\u003c/em\u003e in collided or coupled modes, that depend on the spacer length of the linking mRNA transcript and the recruitment of transcription factors NusG and NusA that can bridge the interface of the RNAP-70S to form an assembly (Webster et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Wang et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Kohler et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Qureshi and Duss, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe occurrence of TTC likely depends on relative rates of transcription and translation, and the frequency of transcription pausing by the RNAP. After transcription initiation, mRNA transcript elongation is highly processive but discontinuous, with a strictly regulated balance between elongation and transcription pausing (Artsimovitch and Landick, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The nascent mRNA itself can directly regulate sequence-dependent transcriptional pausing without requirement for any co-factors (Chan and Landick, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Dey et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Transcript elongation of many genes is paused within the first 200 nts which serves early regulation mechanisms including the recruitment of the transcription factor NusG that facilitates TTC formation (Mooney et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Imashimizu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Hatoum and Roberts, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Washburn et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Promotor-proximal pausing could provide the window of opportunity required to complete translation initiation, which occurs on a timescale of (10\u0026ndash;30) seconds (Shaham and Tuller, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Irastortza-Olaziregi and Amster-Choder, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zhu et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, it remains unclear whether the establishment of TTC occurs only once the pioneering 70S ribosome catches up and collides with the paused RNAP, which may have detrimental effects on transcription fidelity (Wee et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Recent structural evidence shows that RNAP can recruit the 30S ribosomal subunit through transcription factor NusG, potentially as an early step of translation initiation (Webster et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, we have prepared \u003cem\u003eEscherichia coli\u003c/em\u003e 30S translation initiation complexes (30S ICs), including initiation factors IF1, IF2 and IF3 and initiator formyl methionyl-tRNA, and solved cryo-electron microscopy (cryo-EM) structures of the 30S IC in two states. We further assembled 30S ICs on reconstituted transcription elongation complexes (TEC) with a long nascent mRNA that mimics a state of promoter-proximal transcription pausing. Cryo-EM single-particle analysis shows that NusG establishes a physical link between the 30S IC and the paused RNAP, despite the long (\u0026gt;\u0026thinsp;70 nts) mRNA spacer and at \u003cem\u003ein vitro\u003c/em\u003e concentrations far below the proposed dissociation constant of the NusE-NusG interaction (Burmann et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The physically coupled complex of paused transcription and translation initiation, or \u0026lsquo;early expressome\u0026rsquo;, may represent a transient precursor to mature expressomes \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWe explore if the early expressome can be the target for RNA-mediated regulation by testing if the 30S IC-recruited transcript can be accessed by small regulatory RNA (sRNA) that represses the expression of the target gene through base-pairing complementarity with the mRNA. We used the sRNA MicC that directs cleavage and turnover of the \u003cem\u003eompD\u003c/em\u003e transcript encoding the outer membrane protein OmpD in \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium. Assisted by the RNA chaperone Hfq, MicC recognizes \u003cem\u003eompD\u003c/em\u003e and induces downstream \u003cem\u003eompD\u003c/em\u003e-cleavage by the endoribonuclease RNase E (Pfeiffer et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Bandyra et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Considering that \u003cem\u003eompD\u003c/em\u003e-targeting occurs in the coding region (codons 23\u0026ndash;26) and that the ternary complex of MicC and Hfq on \u003cem\u003eompD\u003c/em\u003e does not prevent ribosome engagement or translocation (Pfeiffer et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), the 30S IC and a TEC paused immediately downstream of the recognition sequence for MicC could potentially be targets for riboregulation. We observe that the \u003cem\u003eompD\u003c/em\u003e transcript in the early expressome can be cleaved by the endoribonuclease RNase E within the multi-enzyme RNA degradosome at a defined site that is directed by the MicC sRNA. Although the rate of mRNA degradation is slower when the target transcript lies within the early expressome, the preference of RNase E for the sRNA-guided cleavage site over alternative, unspecific cleavage positions is increased, suggesting that the early expressome may help expose the seed pairing region in the mRNA for recognition by sRNA and Hfq. Our cryo-EM data indicates that the flexible tether between RNAP and 30S ribosomal subunit can allow the early expressome to accommodate transcript recognition and degradation close to the RNAP. Thus, based on evidence presented here and from other studies, we propose that the establishment of TTC may serve as a transient checkpoint for the nascent mRNA intermediate which, once tagged by sRNA, becomes the target for co-transcriptional degradation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCryo-EM structure of\u003c/strong\u003e \u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003e30S translation initiation complexes on the\u003c/strong\u003e \u003cstrong\u003eompD\u003c/strong\u003e \u003cstrong\u003emRNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe had previously prepared 30S IC on a 187 nts long fragment of the \u003cem\u003eompD\u003c/em\u003e transcript encompassing the 5\u0026apos;-UTR from position \u0026minus;\u0026thinsp;69 up to positions\u0026thinsp;+\u0026thinsp;118 (with +\u0026thinsp;1 being A in the AUG start codon) and shown that the \u003cem\u003eompD\u003c/em\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e-30S IC\u003c/span\u003e can be recognized by MicC-Hfq and targeted for deactivating cleavage at position\u0026thinsp;+\u0026thinsp;83 by the RNA degradosome (Bandyra et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Here, we again reconstituted 30S IC on \u003cem\u003eompD\u003c/em\u003e together with formyl methionyl-tRNA and initiation factors IF1, IF2 and IF3, and determined their structure by cryo-EM single particle analysis. The image processing resulted in two distinct structures of the \u003cem\u003eompD\u003c/em\u003e-30S IC that have either IF3 or initiator tRNA and IF2 recruited (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cstrong\u003eMethods, Suppl. Figure\u0026nbsp;1, Table\u0026nbsp;1\u003c/strong\u003e). Both structures contain well-defined density for the 30S ribosomal subunit, as well as IF1 and the mRNA which are recruited in previously described positions (Hussain et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The mRNA is bound to the cavity that is formed between head and body regions of the 30S, and the Shine-Dalgarno sequence (SD) in the 5\u0026apos;-UTR forms a duplex with the anti-SD of the 16S rRNA. IF3 is bound at the 30S near IF1 and mRNA (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA,B) in an orientation corresponds with a previously reported model of the \u003cem\u003eT. thermophilus\u003c/em\u003e 30S IC in \u0026lsquo;state 1\u0026rsquo; (Hussain et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). This arrangement also agrees with a recent report of \u003cem\u003eE. coli\u003c/em\u003e IF3-bound 30S in which the IF3-CTD is positioned at the P-site (Uday et al., 2023).\u003c/p\u003e\n\u003cp\u003eOur second structure, which arose from the same specimen but was reconstructed from a separate particle subset, lacks IF3 but contains IF2 and the initiator tRNA in addition to IF1 and the 24 nts segment of the \u003cem\u003eompD\u003c/em\u003e mRNA (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). The start codon of the mRNA forms triplet base pairs with the anticodon of the bound initiator tRNA that is bound in the P-site (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD), confirming that the 30S IC is located at the +\u0026thinsp;1 position of the \u003cem\u003eompD\u003c/em\u003e mRNA fragment. The arrangement of the IC components on the 30S ribosomal subunit is consistent with previously reported models of the \u003cem\u003eT. thermophilus\u003c/em\u003e 30S IC in \u0026lsquo;state 4\u0026rsquo; (Hussain et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, our cryo-EM density resolves the N2 subdomain of the \u003cem\u003eE. coli\u003c/em\u003e IF2 (residues 295\u0026ndash;370) which attach to helix 16 of the 16S rRNA, resembling reported structures of \u003cem\u003eP. aeruginosa\u003c/em\u003e IF2 at the 70S-IC (Basu et al., 2022) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). The head region of the 30S ribosomal subunit displays a lower degree of mobility in respect to the 30S body, compared to the structure that contains IF3 but lacks the initiator tRNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRibosomal S1 protein contacts the 5\u003c/strong\u003e\u0026apos;\u003cstrong\u003e-UTR at the shoulder of the 30S\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe observe additional cryo-EM density at the 5\u0026apos; mRNA exit site that allows unambiguous placement of the first two domains of S1 (residues 5-171) in an orientation that is in agreement with previous observations (D\u0026apos;Urso et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC \u003cstrong\u003eright, F\u003c/strong\u003e). Notably, the constellation of the S1 domains differs from available 70S ribosome and expressome structures (Webster et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e, Wang et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e, Loveland and Korostelev, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e), but agrees with reports of the 30S-tRNA-RNAP complex (Webster et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e), suggesting that S1 undergoes large conformational changes during translation initiation.\u003c/p\u003e\n\u003cp\u003eAt lower map-thresholds the density for S1 in our \u003cem\u003eompD\u003c/em\u003e-30S IC structure further extends from the described binding site along the shoulder of the 30S body towards proteins S6 and S11 and up to the binding site of IF3. The predicted model for the S1 protein in isolation (Varadi et al., 2022) shows an array of six OB fold-like S1-domains. The observed density in our 30S IC can accommodate additional S1-domains in an elongated arrangement and along with potentially bound 5\u0026apos;-UTR of the mRNA. This is consistent with reports of S1 interacting with upstream portions of mRNA on the 30S surface (Sengupta et al., 2001) and/or binding of A/U-rich segments that precede the Shine-Dalgarno sequence (Komarova et al., \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn a paused\u003c/strong\u003e \u003cstrong\u003eompD\u003c/strong\u003e \u003cstrong\u003etranscription elongation complex, the seed pairing region remains accessible to sRNA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA polymerase can transiently pause during transcription. Genome-wide studies of \u003cem\u003ein vivo\u003c/em\u003e transcriptional pausing in \u003cem\u003eE. coli\u003c/em\u003e have identified a gene-encoded signal element with the consensus motif G\u003csub\u003e\u0026minus;\u0026thinsp;10\u003c/sub\u003eY\u003csub\u003e\u0026minus;1\u003c/sub\u003eG\u003csub\u003e+\u0026thinsp;1\u003c/sub\u003e (where \u0026minus;\u0026thinsp;1 corresponds to the 3\u0026apos;-end of the nascent RNA, with Y representing a pyrimidine base, \u003cstrong\u003eSuppl. Figure\u0026nbsp;2A\u003c/strong\u003e) at which transcriptional pausing occurs with increased likelihood (Vvedenskaya et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e, Larson et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e, Imashimizu et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). With high conservation, the \u003cem\u003eompD\u003c/em\u003e gene harbours this sequence motif in its coding region, 28 nts downstream of the recognition sequence for the sRNA MicC (\u003cstrong\u003eSuppl. Figure\u0026nbsp;2B\u003c/strong\u003e). Two adjacent guanine bases at the upstream end (positions G\u003csub\u003e\u0026minus;\u0026thinsp;9\u003c/sub\u003e and G\u003csub\u003e\u0026minus;\u0026thinsp;10\u003c/sub\u003e within the motif) could stabilize the DNA-RNA hybrid of a paused TEC in pre- and post-translocated states (Imashimizu et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). In \u003cem\u003eS.\u003c/em\u003e Typhimurium \u003cem\u003eompD\u003c/em\u003e, the G\u003csub\u003e+\u0026thinsp;1\u003c/sub\u003e nucleotide of the pause motif lies at position\u0026thinsp;+\u0026thinsp;106 in the coding region and is consistent with a putative paused transcript species of corresponding length that we observed when transcribing \u003cem\u003eompD in vitro\u003c/em\u003e using \u003cem\u003eE. coli\u003c/em\u003e RNAP (\u003cstrong\u003eSuppl. Figure\u0026nbsp;2C-E\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo investigate whether transcription pausing at the described position impacts \u003cem\u003eompD\u003c/em\u003e recognition by Hfq/MicC and deactivating cleavage by the RNA degradosome, we reconstituted TECs \u003cem\u003ein vitro\u003c/em\u003e with \u003cem\u003eompD\u003c/em\u003e segments encompassing the 5\u0026apos;-UTR (from position \u0026minus;\u0026thinsp;69) up to positions\u0026thinsp;+\u0026thinsp;105 or +\u0026thinsp;99 (\u003cstrong\u003eSuppl. Figure\u0026nbsp;2F\u003c/strong\u003e). The 9 terminal nucleotides at the 3\u0026apos;-ends were altered to match the template DNA strand of a short, artificial transcription bubble for TEC reconstitution (\u003cstrong\u003eSuppl. Figure\u0026nbsp;2G\u003c/strong\u003e). We validated correct \u003cem\u003eompD\u003c/em\u003e-TEC assembly by cryo-EM (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-C, \u003cstrong\u003eSuppl. Figure\u0026nbsp;2H-L\u003c/strong\u003e). The 3D reconstruction shows clear density for two \u0026alpha;, \u0026beta; and \u0026beta;\u0026rsquo; subunits of the RNAP core enzyme, but none for the \u0026omega; subunit (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). As expected, the substrate binding site of the RNAP harbours the duplex DNA construct, which can be continuously traced through the cryo-EM density. The transcription bubble displays the melted base pairs of which 10 nucleotides in the template DNA are hybridised to the 3\u0026apos;-end of the mRNA (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). The active site harbours an unpaired template nucleobase with RNAP in the post-translocated state. Though present in the sample and co-eluted from preparative size exclusion chromatography performed before sample vitrification, Hfq and MicC are not resolved in the cryo-EM map. Nevertheless, the structure shows that the \u003cem\u003eompD\u003c/em\u003e-TEC assembles as anticipated and demonstrates that the presence of MicC-Hfq does not disturb the complex.\u003c/p\u003e\n\u003cp\u003eThe reconstituted \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC places the RNAP at the above-described putative pause element, 28 nts downstream of the MicC hybridisation site. Based on our structure and \u003cem\u003ein silico\u003c/em\u003e docking with available molecular models of TEC (PDB: 2ppb) and RNase E (PDB: 2c0b) (Vassylyev et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e, Callaghan et al., \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e), a reconstituted \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC would expose the cleavage site at position\u0026thinsp;+\u0026thinsp;83 with six downstream nucleotides outside the RNAP exit channel and should thus be accessible to the active centre of RNase E. Conversely, a six nucleotides shorter \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e99\u003c/em\u003e\u003c/sub\u003e-TEC should still allow the MicC seed region to hybridize to the recognition sequence in \u003cem\u003eompD\u003c/em\u003e, but the +\u0026thinsp;83-cleavage site would be masked by the RNAP exit channel and therefore sterically protected from RNase E cleavage. Indeed, \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e99\u003c/em\u003e\u003c/sub\u003e-TECs were cleaved by RNase E at the MicC-induced\u0026thinsp;+\u0026thinsp;83 cleavage site much less efficiently than free \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e99\u003c/em\u003e\u003c/sub\u003e in the absence of RNAP (\u003cstrong\u003eSuppl. Figure\u0026nbsp;2M, N\u003c/strong\u003e), indicating correct TEC assembly at the 3\u0026apos;-end of the mRNA and the stability of the complex throughout exposure to regulatory RNA, Hfq and RNase E. The \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC on the other hand remains accessible to MicC-induced cleavage at position\u0026thinsp;+\u0026thinsp;83 by RNase E (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE) and the full RNA degradosome complex (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). Moreover, cleavage at position\u0026thinsp;+\u0026thinsp;83 appears more efficient when \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e is a part of elongation complex, indicated by an increased rate of accumulation of the corresponding intermediate (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). The +\u0026thinsp;72-cleavage product is an off-target species that is only observed \u003cem\u003ein vitro\u003c/em\u003e (Pfeiffer et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e, Bandyra et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e, Bandyra et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCryo-EM structure of a coupled transcription elongation-translation initiation complex\u003c/h2\u003e\n \u003cp\u003ePromoter-proximal pausing of transcription in the coding region of genes after synthesis of the ribosome binding site could allow the translational and transcriptional machineries to synchronize. We explored whether the paused \u003cem\u003eompD\u003c/em\u003e-TEC could engage in physical coupling with the pioneering 30S ribosomal subunit during recruitment of translation initiation factors. As described above, we have successfully reconstituted TECs and 30S ICs separately and verified that the \u003cem\u003eompD\u003c/em\u003e-30S IC assembles at the start codon and demonstrated that the \u003cem\u003eompD\u003c/em\u003e-TEC assembles on the artificial transcription bubble at the 3\u0026apos;-end of the \u003cem\u003eompD\u003c/em\u003e-mRNA fragment, mimicking paused transcription elongation complexes. We then reconstituted both assemblies together on the \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e transcript fragment in the presence of transcription factors NusA and NusG and performed cryo-EM analysis (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cstrong\u003eSuppl. Figure\u0026nbsp;3\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eThrough iterative rounds of 3D classification, we selected 30S particles that showed clear signal for the initiation factors and initiator tRNA (\u003cstrong\u003eSuppl. Figure\u0026nbsp;3\u003c/strong\u003e). The resulting map can accommodate the 30S IC, mRNA, initiator tRNA and IF1-3 in the positions that we observe in our 30S IC data, and we again observe the elongated density near the mRNA exit site that can be accounted for by the ribosomal S1 protein (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eAdditionally, our map displays a large area of unoccupied density on top of the 30S head which is not present in the \u003cem\u003eompD\u003c/em\u003e-30S IC structures. To improve the map in this area, we subtracted the signal for the 30S IC from the cryo-EM particles and performed iterative rounds of 3D refinement and classification, which enabled the isolation of a subset of particles that refined to fit the TEC portion of the coupled complex to ~\u0026thinsp;6 \u0026Aring; resolution (\u003cstrong\u003eSuppl. Figure\u0026nbsp;3\u003c/strong\u003e). The 30S-coupled TEC resembles the above-described structure of the \u003cem\u003eompD\u003c/em\u003e-TEC that was solved in the absence of the 30S IC but displays additional density for the \u0026omega; subunit of RNAP as well as for the N-terminal domain (NTD) of transcription factor NusG. The NusG NTD is bound at the previously described binding site between b and b\u0026apos; subunits near the unwound non-template DNA strand (Kang et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). We also observe well-defined density for the NusG-CTD on the 30S ribosomal subunit near NusE (small ribosomal subunit protein S10) (Burmann et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e), and this density is absent in our structures of the 30S IC that was prepared in the absence of NusG.\u003c/p\u003e\n \u003cp\u003eNotably, density for the TEC appeared only in 3D-reconstructions generated from the subset of 30S particles that also contained signal for the initiation factors and initiator tRNA. Additionally, \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC-IC samples prepared in the absence of NusG did not display physical coupling between TEC and 30S IC (\u003cstrong\u003eSuppl. Figure\u0026nbsp;4\u003c/strong\u003e), suggesting that the physical coupling between RNAP and 30S IC is dependent both on NusG and mRNA-engagement by the small ribosomal subunit.\u003c/p\u003e\n \u003cp\u003eWhen the cryo-EM particles of the final subset are aligned on either 30S IC or RNAP, the respective other component remains only visible as noisy density (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, D; \u003cstrong\u003eSuppl. Figure\u0026nbsp;3\u003c/strong\u003e). This high mobility between TEC and 30S IC within the early expressome likely results from the long mRNA linker in which the start codon at the P-site of the 30S IC and the 3\u0026apos;end at the TEC lie almost 90 nucleotides apart. Nevertheless, the general orientation between TEC and 30S IC is consistent with the \u0026lsquo;coupled\u0026rsquo; state that was described for expressomes with mRNA linkers of 8\u0026ndash;10 codons, while complexes with shorter mRNA linkers position the TEC closer to the 70S mRNA entry site in a \u0026lsquo;collided\u0026rsquo; complex that is incompatible with NusG recruitment (Wang et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e, Webster et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e, Kohler et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eOverall, our structural data demonstrates that a putative early expressome can be formed \u003cem\u003ein vitro\u003c/em\u003e from a TEC and a 30S IC, and the physical coupling of the two subcomplexes relies on NusG which forms a link between mRNA-engaged 30S and RNAP.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eThe coupled transcription elongation-translation initiation complex can be accessed at a target site by sRNA for effector recruitment\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe linker mRNA in the early expressome model loops out between the TEC and 30S IC, which may provide an opportunity for sRNA pairing to a target site. To test whether the \u003cem\u003eompD\u003c/em\u003e-mRNA, when embedded in the TEC-IC, is still accessible for recognition by MicC and deactivating cleavage by the RNA degradosome, we performed degradation reactions with the RNA degradosome in the presence of 5\u0026apos;P-MicC and Hfq (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cstrong\u003eSuppl. Figure\u0026nbsp;5)\u003c/strong\u003e. Alongside, we also subjected the free \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e segment as well as the individual sub-assemblies \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC and \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-30S IC to degradation reactions. The degradation rates of \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e decreased with rising complexity of the assembly, down to a 4-fold slower degradation of NusG-containing \u003cem\u003eompD\u003c/em\u003e-TEC-IC compared to free \u003cem\u003eompD\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Double-exponential equation fitting of the formation of the +\u0026thinsp;83-cleavage intermediate and degradation of \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e is shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, C. The formation rate allows comparison of cleavage specificity for the +\u0026thinsp;83 site which is expressed as the formation rate of the +\u0026thinsp;83 intermediate over the degradation rate of the \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e starting material. Cleavage specificities for the +\u0026thinsp;83 site in the different \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e assemblies are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD. The RNA degradosome shows the lowest preference for +\u0026thinsp;83-cleavage on free \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e and the highest on the \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC-IC substrate. The fraction of the unspecific\u0026thinsp;+\u0026thinsp;72-cleavage intermediates on the other hand is highest in the case of free \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e, reduced for the \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC and -IC and was unquantifiable in the early expressome (\u003cstrong\u003eSuppl. Figure\u0026nbsp;5\u003c/strong\u003e). The observation that MicC-guided processing by RNase E occurs with higher specificity when RNAP and/or 30S IC are engaged with the \u003cem\u003eompD\u003c/em\u003e mRNA suggests that the gene expression machineries might facilitate the recognition of \u003cem\u003eompD\u003c/em\u003e by the sRNA and/or present the MicC-\u003cem\u003eompD\u003c/em\u003e hybrid in a way that increases susceptibility to attacks by the RNA degradosome at the +\u0026thinsp;83cleavage site.\u003c/p\u003e\n \u003cp\u003eThe cryo-EM maps of the \u003cem\u003eompD\u003c/em\u003e\u003csub\u003e\u003cem\u003e105\u003c/em\u003e\u003c/sub\u003e-TEC-IC complex reveal conformational sub-states that may facilitate regulatory access of the sRNA to the seed pairing site in the transcript (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). One of the modes of conformational variation is in a relative tilting of the RNAP with respect the 30S IC, and this is correlated with additional density on the exposed segment of the mRNA (\u003cstrong\u003eright panel\u003c/strong\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cstrong\u003e)\u003c/strong\u003e. The density is not sufficiently resolved to build a detailed model, but the shape of the envelope can accommodate Hfq and may represent a transient species in which the sRNA/Hfq interacts with the exposed seed region. The schematic in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF illustrates a potential model for this transient access.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we have explored the accessibility of a small RNA pairing site in a target transcript at early stages of transcription elongation and translation initiation. We reconstituted transcription elongation complexes (TEC) mimicking a state of promoter-proximal transcription pausing of the target mRNA and assembled the 30S translation initiation complex (30S IC) on those mRNAs. Cryo-EM single-particle analysis shows that the transcription factor NusG can physically link the 30S IC and the paused TEC, despite the lengthy mRNA linker (\u0026gt;\u0026thinsp;70 nts) and the \u003cem\u003ein vitro\u003c/em\u003e concentrations used that are far below the proposed dissociation constant of the NusE-NusG interaction (Burmann et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The physically coupled assembly, which we refer to as an \u0026lsquo;early expressome\u0026rsquo;, may represent a transient precursor to mature expressomes \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eDuring the preparation of this manuscript, an independent study reported the \u003cem\u003ein vitro\u003c/em\u003e reconstitution of similar transcription elongation-translation pre-initiation complexes (Webster et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Webster et al. (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) present structures of mRNA delivery complexes between a paused TEC and 30S ribosomal subunit in the inactive states and propose two pathways that initiate the coupling between transcription and translation through transcription-assisted recruitment of mRNA to the ribosome (Webster et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). One pathway involves ribosomal protein S1 of the 30S particle interacting with the TEC and binding the nascent mRNA to form an intermediate or standby complex that directs the nascent transcript to the Shine-Dalgarno interaction site of the 30S. The RNAP is transiently located near the cluster of S1 OB-domains and mRNA exit site of the 30S and subsequently repositioned to be near the mRNA entry site in the translational 30S IC. The second pathway involves RNAP directly binding near the mRNA entry site of the inactive 30S, which then transitions to the active form in the assembled 30S IC by folding the 16S rRNA helix 44 away from the mRNA exit channel into its active position on the 30S body (Webster et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our data include IF1-3 and present the 30S IC in \u0026lsquo;accommodated\u0026rsquo; active states which can be formed via both proposed pathways.\u003c/p\u003e \u003cp\u003eThe structural data presented here also corroborate how the ribosomal S1 might be poised to facilitate interactions with 5'-UTR elements. Webster et al. (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) report that the S1 protein not only facilitates mRNA delivery to selectively accelerate duplex formation with the anti-Shine-Dalgarno sequence but helps RNAP to stimulate translation initiation (Webster et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). S1 assists the remodelling of mRNA secondary structures \u003cem\u003ein vitro\u003c/em\u003e to aid the 30S with mRNA loading and 30S IC formation (Duval et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Kolb et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). When the \u003cem\u003eompD\u003c/em\u003e mRNA is recruited to the 30S IC, RNase E relies on the assembly of the RNA degradosome for access and efficient MicC-guided degradation of the \u003cem\u003eompD\u003c/em\u003e transcript (Bandyra et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Ribosomal S1 has been shown to form direct interaction with several sRNAs from \u003cem\u003eE. coli\u003c/em\u003e (Windbichler et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), thereby possibly exerting the 30S IC\u0026rsquo;s influence on Hfq-MicC-guided RNase E attack while located on the 30S ribosome. Further, direct interactions with the TEC (Sukhodolets and Garges, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) might also facilitate riboregulation at the early stage of nascent transcript recruitment to the translation machinery. The paused RNAP may increase the recognition efficiency of the target element within \u003cem\u003eompD\u003c/em\u003e by Hfq-MicC, consistent with our observation that \u003cem\u003eompD\u003c/em\u003e-cleavage by RNase E is more site-specific at the paused TEC. This is supported by a recent single-molecule study showing that co-transcriptional target recognition by sRNA and Hfq close to the RNAP exit channel is more efficient than recognition post transcription (Rodgers et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The biogenesis of 3'-UTR-derived sRNAs that autoregulate their genes (Hoyos et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), along with kinetic modelling studies, provide further support for mechanisms of co-transcriptional regulation by sRNAs (Reyer et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Access to decay machinery during transcription is also supported by observations of co-transcriptional stabilisation of riboswitches against ribonuclease action (Lou and Woodson, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, a recent study presents a model of co-transcriptional endonucleolytic cleavage of RNA in archaea that leads to transcription termination (You et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), suggesting the possibility of a functionally analogous system in that domain of life. In the early expressome of the \u003cem\u003eompD\u003c/em\u003e transcript in which the RNAP is paused at position\u0026thinsp;+\u0026thinsp;106, the deactivating cleavage site at position\u0026thinsp;+\u0026thinsp;83 for RNase E that is preceded by the recognition site for the MicC sRNA, remains accessible to regulatory RNA and deactivating cleavage by the RNA degradosome in \u003cem\u003ein vitro\u003c/em\u003e reactions.\u003c/p\u003e \u003cp\u003eThe cellular location of the degradosome must be considered to explain how access might be gained to the putative TEC-30S IC complex tagged with a small RNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The envisaged recognition event, in which sRNA recruits degradosome to the 30S IC or TEC-IC complex, could occur in species such as \u003cem\u003eCaulobacter crescentus\u003c/em\u003e, in which the RNA degradative machinery can be associated with the nucleoid or ribonucleoprotein condensates in the cell interior and could have access to nascent mRNA targets (Kim et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). However, in species such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eSalmonella\u003c/em\u003e where the degradosome is compartmentalized to the cytoplasmic membrane, and potentially distant from the nucleoid (Carpousis et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the sRNA-tagged early expressome might be the target for other effectors, such as Rho RNA translocase that terminates transcription through allosteric manipulation of the elongation complex (Molodtsov et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Said et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Hao et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Reyer et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Rho can act to trigger premature transcription termination in the absence of transcription-translation coupling (Wang and Artsimovitch, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Kim et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and its activity on nascent mRNA can be modulated by sRNA (Sedlyarova et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Transcription-terminated transcripts could then encounter the degradosome on the membrane upon their diffusion following the release from RNAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei\u003cb\u003ei\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAnother scenario in which membrane-bound RNase E can act early on nascent transcripts is if the transcriptional-translational machinery is brought close to the membrane, as occurs in the \u0026lsquo;transertion\u0026rsquo; mechanism which was described for \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and suggested to occur also in \u003cem\u003eE. coli\u003c/em\u003e (Woldringh, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Bakshi et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Fishov and Norris, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Bakshi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Roggiani and Goulian, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Kaval et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Transertion, a coupling of transcription, translation and membrane insertion at the membrane component of the type III secretion system, is a process suggested to be common for bacterial membrane proteins. Evidence indicates that membrane-associated RNase E can act on transcripts encoding membrane proteins in \u003cem\u003eE. coli\u003c/em\u003e to result in co-transcriptional degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u003cb\u003eiii\u003c/b\u003e) (Kim et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA fourth scenario in which we envisage RNase E to act on a mRNA-30S IC complex is if the leading edge of a polyribosome comes into proximity of the membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u003cb\u003eiv\u003c/b\u003e). Earlier studies have shown that RNase E and the degradosome can interact with polyribosomes \u003cem\u003ein vitro\u003c/em\u003e and potentially \u003cem\u003ein vivo\u003c/em\u003e, and it was proposed that this forms a passive complex that does not cleave the RNA until activated by a signal, such as a cognate sRNA (Tsai et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In species without RNase E, similar processes are likely to occur, such as in the model firmicute species \u003cem\u003eBacillus subtilis\u003c/em\u003e, where the membrane-bound degradosome is based on the distinct ribonuclease RNase Y (Laalami et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This mode of degradation would enable the co-translational decay of a transcript. It also represents a more economical means of exiting translation, because it avoids generating transcript fragments that lack stop codons and entail the hidden metabolic costs of using the tmRNA system for rescue recovery. The above mechanisms could account for processes of co-translational decay (Huch et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Herzel et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) analogous to the recruitment of the 5'-to-3' exoribonuclease Xrn1 which follows the terminal translating ribosome identified in yeast and other eukaryotic species (Pelechano et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Tesina et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eProtein production\u003c/h2\u003e \u003cp\u003eWild-type 30S subunits were prepared from \u003cem\u003eE. coli\u003c/em\u003e MRE600 strain using zonal centrifugation (Rodnina et al., 1994, Rodnina and Wintermeyer, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Initiator tRNA (fMet-tRNAfMet) and \u003cem\u003eE. coli\u003c/em\u003e initiation factors IF1, IF2, and IF3 were purified according to published protocols (Milon et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). \u003cem\u003eE. coli\u003c/em\u003e Hfq was purified as described (Dendooven et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The RNA degradosome was prepared as described (Dendooven et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eE. coli\u003c/em\u003e core RNAP was expressed from pVS10 plasmid coding for all five subunits (Svetlov and Artsimovitch, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and purified as described (Castro-Roa and Zenkin, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA Production\u003c/h3\u003e\n\u003cp\u003eRNAs were prepared by \u003cem\u003ein vitro\u003c/em\u003e transcription (IVT) as described (Bandyra et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In brief, IVT templates were amplified by PCR from plasmid pVP042-3 that carries the \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium \u003cem\u003eompD\u003c/em\u003e gene (Pfeiffer et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The forward primer introduces a T7 promoter sequence upstream of the transcription initiation site and, where indicated, the reverse primer encodes a non-canonical 9 nts sequence (CGGCGCUGG) at the 3\u0026rsquo; end of the corresponding transcript. IVT reactions containing 3 \u0026micro;g of template DNA, 5 mM of each of ATP, UTP, GTP and CTP, 10 mM DTT and 0.5 U/\u0026micro;L RNaseOUT\u0026trade; (Invitrogen) were incubated with recombinant T7 RNA polymerase in 40 mM Tris, pH 8.0, 25 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM spermidine at 37\u0026deg;C for 5 h. IVT products were DNase I-treated, resolved by 4% denaturing PAGE and excised RNA bands were electroeluted using Elutrap\u0026trade; Electroelution System Kit (Whatman), followed by clean-up using PureLink\u0026trade; RNA Micro Kit (Invitrogen).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssembly of transcription elongation complexes\u003c/h2\u003e \u003cp\u003eTECs were assembled following a previously described strategy (Said et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To anneal nucleic acid scaffolds, 8 \u0026micro;M mRNA fragment was mixed with template DNA (tDNA) in a 1:1.25 molar ratio in 10 mM TRIS, pH 7.6, 40 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, heated to 95\u0026deg;C for 2 min and slowly equilibrated to 37\u0026deg;C. Two volumes TEC reconstitution buffer (20 mM TRIS, pH 7.6, 120 mM potassium acetate, 5 mM magnesium acetate, 1 mM TCEP) were pre-heated to 37\u0026deg;C and added to annealed RNA/tDNA. \u003cem\u003eE. coli\u003c/em\u003e RNAP was added in 1:1 ratio to RNA, followed by 10 min incubation. Non-template DNA was added in 1:1 ratio to tDNA and followed by incubation for 10 min at 37\u0026deg;C. Assembled TECs were prepared fresh and used immediately for degradation assays or assembly of higher order complexes. For cryo-EM specimen preparation, 5 \u0026micro;M TEC was mixed with equimolar amount of pre-incubated (32\u0026deg;C for 30 min) MicC and Hfq injected onto a 3.2/300 Superose 6 size-exclusion chromatography column equilibrated with 20 mM TRIS-Cl, pH 7.6, 120 mM potassium acetate, 5 mM magnesium acetate, 2 mM DTT, 10 \u0026micro;M ZnCl\u003csub\u003e2\u003c/sub\u003e. Fractions containing all components as determined by urea-PAGE and SDS-PAGE were pooled, concentrated to 40 \u0026micro;L and used directly for vitrification.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cb\u003eompD\u003c/b\u003e\u003cb\u003e-30S IC and early expressomes\u003c/b\u003e\u003c/p\u003e \u003cp\u003e30S IC was prepared following a previously described strategy (Goyal et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In brief, 30S subunits were incubated in buffer TAKM\u003csub\u003e20\u003c/sub\u003e (50 mM Tris-HCl [pH 7.5], 70 mM NH\u003csub\u003e4\u003c/sub\u003eCl, 30 mM KCl, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e) for 30 min at 37\u0026deg;C for reactivation. Reactivated 30S subunits were incubated with a 2.5-fold excess of mRNA, 2-fold excess of initiation factors 1\u0026ndash;3 and a 2.5-fold excess of initiator fMet-tRNA\u003csup\u003efMet\u003c/sup\u003e (hereafter tRNA) in the presence of 250 \u0026micro;M GTP or GTPγS (Jena Bioscience) in TAKM\u003csub\u003e7\u003c/sub\u003e buffer (Goyal et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For transcription/translation-coupled complexes, the 30S IC was prepared as described above using pre-assembled \u003cem\u003eompD\u003c/em\u003e-TEC instead of free mRNA and omitting the chromatography step. Coupled TEC-IC for cryo-EM analysis was prepared with final concentrations 0.3 \u0026micro;M 30S, 1.2 \u0026micro;M TEC, 0.6 \u0026micro;M of IFs and tRNA, 10 \u0026micro;M NusA, 20 \u0026micro;M NusG and 5 \u0026micro;M MicC/Hfq and directly used for grid preparation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003etranscription\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe first 306 bases of the \u003cem\u003eompD\u003c/em\u003e gene (-69 to +\u0026thinsp;237) were inserted into the pMMB67HE vector between \u003cem\u003etac\u003c/em\u003e promoter and T1 terminator (Furste et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). For \u003cem\u003ein vitro\u003c/em\u003e transcription assays, 15 \u0026micro;L reactions containing 5 nM \u003cem\u003eompD\u003c/em\u003e-pMMB67HE, 0.5 U/\u0026micro;L RNaseOUT\u0026trade; and 0.5 U \u003cem\u003eE. coli\u003c/em\u003e RNA Polymerase Holoenzyme (NEB, M0551S) were pre-incubated in reaction buffer (40 mM Tris-HCl, pH 7.5, 150 mM KCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM DTT, 0.01% Triton X-100\u0026trade;) at 37\u0026deg;C for 5 minutes. Reactions were started by adding ribonucleotide mix to 5 mM, incubated at 37\u0026deg;C for 1 h and quenched by addition of equal volume stop buffer (200 mM Tris-HCl, pH 7.5, 25 mM EDTA, 300 mM NaCl, 2% SDS, 0.5 mg/mL Proteinase K). After proteolysis at 50\u0026deg;C for 1 h, samples were mixed with 0.5 volumes 2x RNA Loading Dye (Thermo Scientific), heated to 95\u0026deg;C for 2 min and separated by urea PAGE on 8% polyacrylamide gels.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGraphene oxide coating and grid preparation\u003c/h3\u003e\n\u003cp\u003eGraphene oxide (GO) coating was prepared by an adaptation of the drop-casting method (Pantelic et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Quantifoil Cu 300 1.2/1.3 grids were glow-discharged on the darker carbon side (PELCO easiGLOW: 15 mA, 0.28 mBar, 2 min). GO solution (Sigma-Aldrich 763705, 2 mg/mL dispersion) was diluted 10-fold in water and centrifuged at 300 xg for 30 s to pellet insoluble GO flakes. The supernatant was further diluted 10-fold to a working concentration of 0.02 mg/mL. 1 \u0026micro;L working solution was applied to the glow-discharged side of the Quantifoil grids and dried at room temperature for 10 min. GO-coated grids were kept at room temperature for at least 16 h and then directly used for sample vitrification. Quantifoil 1.2/1.3 grids with a 2 nm amorphous carbon support layer were briefly glow-discharged (PELCO easiGLOW: 10 s, 25 mA, 0.39 mBar) directly before sample application. For vitrification, 4 \u0026micro;L of sample was applied to grids and incubated for 30 s. Using a FEI Vitrobot, excess sample was blotted, and grids were plunge-frozen in liquid ethane. Grids were subsequently stored in liquid nitrogen until screening and collection.\u003c/p\u003e\n\u003ch3\u003eCryo-EM data collection and processing\u003c/h3\u003e\n\u003cp\u003eFor structures of \u003cem\u003eompD\u003c/em\u003e-30S IC, 9104 multi-frame movies were collected on a 300 kV FEI Titan Krios equipped with a Gatan K3 detector. For the TEC structure, a dataset of 1619 multi-frame movies was collected on a 300 kV Titan Krios equipped with a Falcon 3 detector in Counting mode. For the TEC-30S IC structure, 12011 multi-frame movies were collected on a 300 kV FEI Titan Krios equipped with a Gatan K3 detector. Details for data collection are summarised in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e. Mult-frame movies were motion-corrected in Relion 3.1 (Scheres, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and the generated micrographs were imported in cryoSPARC v4 (Punjani et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and further processed as illustrated in \u003cb\u003eSuppl. Figures\u0026nbsp;1\u0026ndash;3\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eFor structures of TEC-30S IC assemblies, small datasets were collected on a 200 kV Talos Arctica equipped with a Falcon 3 detector in Counting mode and processed in Relion 3.1 (Scheres, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) as depicted in image processing workflows (\u003cb\u003eSuppl. Figure\u0026nbsp;4B, G, L\u003c/b\u003e).\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available from the PDB and EMDB.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Andrzej Szewczak-Harris and the staff of the Cryo-EM Facility, Dimitri Y. Chirgadze, Steven Hardwick and Lee Cooper, for assistance with data collection. We thank the staff at eBIC for access to facilities and help with data collection. We thank Tom Dendooven, Kathi Frohlich, and Joerg Vogel for helpful comments and suggestions. KJB, BFL and NZ are supported by Wellcome Trust Investigator Awards (222451/Z/21/Z, 200873/Z/16/Z, 217189/Z/19/Z). JJR is supported by a Herchel Smith Studentship. GP is supported by a Benn W Levy\u0026ndash;Vice Chancellor Award SBS DTP PhD studentship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eARTSIMOVITCH, I. \u0026amp; LANDICK, R. 2000. Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals. \u003cem\u003eProc Natl Acad Sci U S A,\u003c/em\u003e 97\u003cstrong\u003e,\u003c/strong\u003e 7090-5.\u003c/li\u003e\n\u003cli\u003eBAKSHI, S., CHOI, H., MONDAL, J. \u0026amp; WEISSHAAR, J. C. 2014. 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Here, we present structural evidence from cryo-EM demonstrating that a bacterial RNA polymerase that is paused proximally to the promoter can associate with the pioneering 30S translation initiation complex (30S IC) through mutual binding of the transcription factor NusG. These findings suggest that the physical link between transcription and translation can be established prior to commitment to protein synthesis. Although the mRNA is embedded in this \u0026lsquo;early expressome\u0026rsquo; complex, it can nonetheless interact with small regulatory RNA (sRNA) and be targeted for cleavage in the protein-coding region by the RNA degradosome assembly \u003cem\u003ein vitro\u003c/em\u003e. The potential tagging of transcripts with sRNA during pioneering and subsequent stages of translation initiation, when the 30S IC is at the 5\u0026prime; end of a polyribosome, may support surveillance processes that ensure efficient and rapid termination of gene expression in response to regulatory signals.\u003c/p\u003e","manuscriptTitle":"Structure of the 30S translation initiation complex coupled to paused RNA polymerase and its potential for riboregulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-11 11:16:59","doi":"10.21203/rs.3.rs-5868712/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5e2e763a-972b-4252-97ba-f3ca5fbc8a22","owner":[],"postedDate":"February 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44071396,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":44071397,"name":"Biological sciences/Molecular biology/RNA metabolism/RNA quality control"}],"tags":[],"updatedAt":"2026-01-21T08:36:07+00:00","versionOfRecord":{"articleIdentity":"rs-5868712","link":"https://doi.org/10.1038/s41467-025-67330-2","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-13 05:00:00","publishedOnDateReadable":"December 13th, 2025"},"versionCreatedAt":"2025-02-11 11:16:59","video":"","vorDoi":"10.1038/s41467-025-67330-2","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67330-2","workflowStages":[]},"version":"v1","identity":"rs-5868712","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5868712","identity":"rs-5868712","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}