{"paper_id":"a1b36a21-9bda-4f9c-8c3c-e87925ad86dc","body_text":"1 \n \nStructure and function of the Si3 insertion integrated into the trigger loop/helix of 1 \ncyanobacterial RNA polymerase 2 \n 3 \nM. Zuhaib Qayyum 1,4,*, Masahiko Imashimizu 1,2,*, Miron Leanca 3,*, Rishi K. Vishwakarma 1,*, 4 \nAmber Riaz-Bradley3, Yulia Yuzenkova3,5 and Katsuhiko S. Murakami1,5 5 \n1Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, 6 \nThe Center for Structural Biology, The Pennsylvania State University, University Park, PA 7 \n16802, USA 8 \n2Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced 9 \nIndustrial Science and Technology, Tsukuba, 305-8565 Japan 10 \n3The Centre for Bacterial Cell Biology, Newcastle University, UK 11 \n4Current address: Protein Technologies Center, Inspiration4 Advanced Research Center, 12 \nDepartment of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, 13 \nUSA 14 \n*Contributed equally to this work 15 \n5To whom correspondence should be addressed: kum14@psu.edu  (K.S.M.), 16 \ny.yuzenkova@ncl.ac.uk (Y.Y.) 17 \n 18 \nAuthor Contributions: Y.Y. and K.S.M. designed the research; M.Z.Q., M.I., M.L., R.K.V. and 19 \nA.R-B performed the research; M.Z.Q., M.I., Y.Y. and K.S.M. analyzed the data; and M.I., Y.Y. 20 \nand K.S.M. wrote the paper. 21 \nCompeting Interest Statement: The authors declare no competing interests. 22 \nClassification: Biological Sciences/Biochemistry 23 \nKeywords: cyanobacteria, RNA polymerase, transcription, cryo-EM 24 \nThis PDF file includes: 25 \nMain Text 26 \nFigures 1 to 5 27 \nSupplemental Figures 1-7, Tables 1-2, Supplemental Movie Legends 1-2 28 \n  29 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n2 \n \nAbstract 30 \nCyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA 31 \npolymerases (RNAPs) with characteristics that distinguish from canonical bacterial RNAPs. The 32 \nlargest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, β ’1 and β ’2, 33 \nand contains the largest known lineage-specific insertion domain, Si3, located in the middle of 34 \nthe trigger loop and spans approximately half of the β ’2 subunit. In this study, we present the X-35 \nray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation 36 \ncomplex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound 37 \nat the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main 38 \nbody of cyRNAP, fits into cavities of cyRNAP and shields the binding site of secondary channel-39 \nbinding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger 40 \nhelix upon iNTP binding at the active site results in a large swing motion of Si3; however, this 41 \ntransition does not affect the catalytic activity of cyRNAP due to its minimal contact with 42 \ncyRNAP, NusG or DNA. This study provides a structural framework for understanding the 43 \nevolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may 44 \nprovide insights into the molecular mechanism of transcription in specific environment of 45 \nphotosynthetic organisms. 46 \n 47 \nSignificance statement: 48 \nCellular RNA polymerase (RNAP) carries out RNA synthesis and proofreading reactions 49 \nutilizing a mobile catalytic domain known as the trigger loop/helix. In cyanobacteria, this 50 \nessential domain acquired a large Si3 insertion during the course of evolution. Despite its 51 \nelongated shape and large swinging motion associated with the transition between the trigger 52 \nloop and helix, Si3 is effectively accommodated within cyRNAP, with no impact on the 53 \nfundamental functions of the trigger loop. Understanding the significance of Si3 in cyanobacteria 54 \nand chloroplasts is expected to reveal unique transcription mechanism in photosynthetic 55 \norganisms. 56 \n 57 \n 58 \n 59 \n  60 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n3 \n \nIntroduction 61 \nCyanobacteria and chloroplasts of algae and higher plants are characterized by oxygen-62 \nevolving photosynthesis and are phylogenetically closely related. These genomes are transcribed 63 \nby a bacterial-type RNA polymerase (cyRNAP and plastid-encoded RNAP, PEP, respectively) 64 \naided by transcription initiation σ factors for recognition of specific promoters (1-3). Although 65 \ncyRNAPs and chloroplast PEPs retain the fundamental functions of bacterial RNAPs, they 66 \npossess several distinct characteristics that distinguish them from canonical bacterial RNAPs. 67 \nFirst, the largest subunit of cyRNAP is separated into two polypeptides, β ’1 and β ’2, 68 \nwhich are encoded by the rpoC1 and rpoC2 genes, respectively (Fig. 1A). In Synechococcus 69 \nelongatus, which is the cyanobacterium used for the cryo-EM structural study of RNAP 70 \ndescribed herein, the 624 residue β ’1 and 1,318 residue β ’2 subunits correspond to the amino 71 \n(N)-terminal one-third and the carboxy (C)-terminal two-thirds of the 1,407 residue β ’ subunit in 72 \nEscherichia coli, respectively. A junction between the β ’1 and β ’2 subunits is positioned before 73 \nthe conserved region E (4, 5). The β ’1 subunit contains the clamp and the catalytic double-psi-β -74 \nbarrel domain coordinating a Mg 2+ ion; the β ’2 subunit contains the rim helix, bridge helix, 75 \ntrigger loop and jaw domain. 76 \n Second, cyRNAP contains the largest known lineage-specific insertion domain, Si3 (645 77 \nresidues), which spans approximately half the size of the β 2’ subunit and is located in the middle 78 \nof the trigger loop (Fig. 1A) (6, 7). The trigger loop plays a central role in nucleotide selection, 79 \nRNA synthesis and RNA cleavage during proofreading by cellular RNAPs (8). In the absence of 80 \nnucleotide triphosphate (NTP) substrate, the tip of the trigger loop is located away from the 81 \nactive site (9). Upon binding of complementary incoming NTP (iNTP) at the active site, the 82 \ntrigger loop folds to form a trigger helix containing two α -helices, which extensively interacts 83 \nwith the base and triphosphate groups of iNTP and facilitates the nucleotidyl transfer reaction 84 \n(8). The Si3 insertion is found in RNAPs of gram-n egative bacteria in the middle of the trigger 85 \nloop (evolutionarily conserved region G; Fig. 1A ). Si3 is composed of repeats of the conserved 86 \nsandwich-barrel hybrid motif (SBHM). Escherichia coli  (E. coli) RNAP contains two copies of 87 \nSBHM (SFig. 1A) (10), and sequence analysis indicates that up to seven copies of SBHM are 88 \npresent in the Si3 insertion of cyRNAP (6). The structure and function of the Si3 insertion in E. 89 \ncoli RNAP have been well characterized; it is involved in stabilizing the open complex and RNA 90 \nhairpin-dependent ( his) and -independent ( ops) transcription pausing (11, 12) and is highly 91 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n4 \n \nmobile, with its confirmation being dependent on the folded/unfolded state of the trigger 92 \nloop/helix and binding of transcription factors (Gre, DksA) at the secondary channel of RNAP 93 \n(13, 14). In addition, structural and functional analyses of Si3 in cyRNAP have recently been 94 \ninitiated. According to the cryo-electron microscopy (cryo-EM) structure of the cyRNAP 95 \npromoter complex (15), Si3 forms an “arch” with region 2 of the σ factor, the element involved 96 \nin opening the DNA duplex at the -10 position of the promoter. This arch stabilizes the promoter 97 \ncomplex, and its removal affects the fitness and stress resistance of cyanobacteria. Notably, the 98 \nSi3-σ contact remains intact upon trigger loop refolding into the trigger helix after iNTP addition 99 \nto the initiation complex with the short RNA transcript. After transition to the elongation phase, 100 \nit is unknown whether Si3 becomes mobile in the presence of transcription elongation factors 101 \nsuch as NusG and how Si3 affects refolding of the trigger helix and the catalytic activity of 102 \nRNAP. 103 \n In this work, we structurally and biochemically analyzed cyRNAP elongation complex 104 \n(EC) to understand the functional importance of Si3 in the elongation phase of transcription. We 105 \nsolved the X-ray crystal structure of Si3 and cryo-EM structures of the cyRNAP EC with NusG 106 \nin the presence and absence of iNTP bound at the active site. 107 \n 108 \nResults 109 \nX-ray crystal structure of Thermosynechococcus elongatus BP-1 Si3 (TelSi3) 110 \n We investigated the structure of the separate Si3 protein of the thermophilic 111 \ncyanobacterium Thermosynechococcus elongatus BP-1  (TelSi3) by X-ray crystallography. The 112 \nDNA sequence encoding Si3 (residues 345-983) was cloned and inserted into a vector for 113 \nexpression in E. coli cells, and the resulting protein was purified to homogeneity. Initial attempts 114 \nto crystallize TelSi3 were unsuccessful. Limited trypsinolysis revealed that the amino-terminal 115 \n(N-terminal) 91 residues of TelSi3 are sensitive to proteolysis (SFig. 2A), indicating flexibility, 116 \nwhich potentially hindered crystallization. We then cloned and expressed TelSi3, which lacks the 117 \nN-terminal 91 residues (TelSi3 ΔN, residues 435 to 983) and thus forms large crystals (Fig. 1B) 118 \nbelonging to the P3(2)21 space group (six TelSi3 ΔN copies per asymmetric unit; Fig. 1C). We 119 \nwere unable to generate a TelSi3 ΔN model suitable for molecular replacement based on the 120 \nprotein sequence (e.g., by SWISS-MODEL; SFig. 2C). Therefore, the experimental phase was 121 \nachieved by the single-wavelength anomalous dispersion (SAD) method using selenomethionine 122 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n5 \n \n(SeMet)-labeled TelSi3ΔN protein (SFig. 2B). The 3.2 Å resolution experimental density map 123 \nallowed us to build the structures of four full-length and two partial models of TelSi3 ΔN in the 124 \nasymmetric unit (STable 1). The AlphaFold (20) structural prediction for TelSi3ΔN was in close 125 \nagreement with the X-ray structure, with an RMSD of 1.08 Å (SFig. 2C). 126 \n TelSi3 ΔN (150 Å in length and 50 Å in width) is longer than the canonical bacterial 127 \nRNAP (e.g., 110 × 130 Å: E. coli RNAP) (SFig. 1B). TelSi3 ΔN comprises seven SBHMs 128 \n(SBHM-2 to SBHM-8). The X-ray crystal structure of the N-terminal region (81 residues) of Si3 129 \nfrom S. elongatus  PCC 7942 (15) showed an independently folded SBHM (SBHM-1). This 130 \nregion corresponds to the 91 N-terminal residues of TelSi3 (missing in the crystallized 131 \nTelSi3ΔN), indicating that cyRNAP contains 8 copies of SBHM within Si3 (Fig. 1C, SFig. 3). 132 \nTelSi3 has a swordfish-shaped profile, with distinct “tail”, “fin”, “body” and “head” 133 \nsubdomains formed by SBHM-1, SBHM-2/8, SBHM-3/4/5 and SBHM-6/7, respectively (Fig. 134 \n1D). Notably, the SBHMs in TelSi3 are not structured in a simple tandem arrangement (Fig. 2D 135 \nand SFig. 3), in contrast to E. coli  Si3, which contains two independently folded SBHMs 136 \nconnected by a short linker (SFig. 1A) (10). Although each SBHM has a core antiparallel β -sheet 137 \ntopology, connections between the β -sheets vary as the polypeptide chain folds over itself (Fig. 138 \n1D). In addition, the sequences of SBHM-1, -6, -7 and -8 are continuous; the others (SBHM-2, -139 \n3, -4 and -5) contain structural elements from distant regions of the polypeptide sequence. We 140 \nassessed conformational flexibility by comparing the four full-length TelSi3ΔN structures from 141 \nthe asymmetric unit using the Si3-fin as a reference for superimposition. This showed substantial 142 \nconformational variation in the Si3- h ead, allo wing for a 24  A/i18  displacement associated with an 143 \n11° rotation (Fig. 1E). 144 \n 145 \nCryo-EM structure of the Synechococcus elongatus RNAP elongation complex with NusG 146 \n To investigate the structure of cyRNAP and the dynamics of Si3 at the elongation stage, 147 \nwe determined the cryo-EM single-particle reconstruction structure of the cyRNAP EC (SFig 4, 148 \nSTable 2). NusG was also included in the EC, as most ECs contain NusG under physiological 149 \ntranscription conditions (16, 17), and physical contact between Si3 and NusG was investigated. 150 \n We used recombinant double affinity-tagged S. elongatus cyRNAP to avoid isolation of 151 \nany chimeric cyRNAP containing E. coli RNAP subunit. EC was assembled by mixing cyRNAP, 152 \nNusG and the DNA/RNA scaffold (Fig. 2A). The preferred particle orientation issue of EC-153 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n6 \n \nNusG was resolved by adding CHAPSO (final concentration of 0.8 mM) to the sample before 154 \napplication to the cryo-EM grid (18) . The cryo -EM s truc t ure was determined  with an  overal l  155 \nreso lution  of 3 A/i18 , revealing well-defined cryo-EM densities for cyRNAP, the N-terminal 156 \ndomain of NusG (residues 19-138) and the DNA/RNA hybrid (Fig. 2B). The densities of the 157 \nsingle-stranded nontemplate DNA in the transcription bubble and the single-stranded RNA 158 \nwithin the RNA exit channel were traceable due to their respective interactions with NusG and 159 \nthe RNA exit channel (Fig. 2C). The carboxyl-terminal (C-terminal) domains of the α  subunits 160 \nand the Kyrpides-Ouzounis-Woese (KOW) domain of NusG were disordered. 161 \n By contacting both the upstream and downstream DNA duplexes, NusG seemed to 162 \nmaintain a 90 ° bend in the DNA centered at the RNAP active site (SFig. 5A), which may 163 \nstabilize the DNA/RNA holding of cyRNAP. To evaluate the role of NusG, we immobilized 164 \nreconstituted ECs on agarose beads and challenged the complex with 300 mM NaCl in the 165 \nabsence of NusG. There was a significant reduction in the proportion of RNA released from the 166 \ncomplex compared with the EC in the presence of NusG (SFig. 5B), indicating its stabilizing 167 \neffect. Notably, compared with its orthologs from E. coli, Bacillus subtilis and Mycobacterium 168 \ntuberculosis, the cyanobacterial NusG gene possesses a longer and more positively charged loop 169 \n(residues 110-122) within the N-terminal domain. This loop extends toward the downstream 170 \nDNA and single-stranded non-template DNA within the transcription bubble (SFig. 5A). 171 \nDeletion of this cyanobacteria-specific loop (NusG Δ110-122) significantly reduced the stabilizing 172 \neffect of NusG (SFig. 5B). 173 \n 174 \nSi3 runs along the cavities of cyRNAP and shields the binding site of DksA/Gre factors 175 \n By fitting the models of RNAP (without Si3), NusG and the DNA/RNA scaffold, we 176 \nelucidated a density corresponding to Si3, which extends starting from the trigger loop and then 177 \nmoves below the rim helix ( β ’2 subunit), running along the lobe/protrusion domains ( β  subunit) 178 \nand nearly reaching the upstream DNA (Fig. 2B, SMovie 1). The overall structure of cyRNAP is 179 \nnearly identical to the structures of other bacterial RNAPs, including those of E. coli  and M. 180 \ntuberculosis (19, 20), indicating that Si3 runs along the cavities of RNAP without influencing its 181 \ngeneral shape or conformation. The crystal structures of Si3 containing both SBHM2-8 and 182 \nSBHM1 were fitted to their corresponding cryo-EM density. The cryo-EM density of the Si3-183 \nhead was weak and had a low resolution (Fig. 2B, SFig. 4E), suggesting its mobility.  184 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n7 \n \n Si3-tail is positioned in front of the rim helix (Fig. 3A). Si3-fin is positioned below the 185 \nrim helix, and the extended SBHM2 loop (residues 463-471) fills a gap between the β ’2 jaw and 186 \nβ  lobe domains. Si3-body is located beside the lobe and protrusion domains of the β  subunit, and 187 \nSi3-head reaches the upstream DNA (Figs. 2B and 3A). Si3-fin contacts the bottom part of the 188 \nrim helix, but only a few amino acid residues of Si3 contact the main body of RNAP and NusG, 189 \nsuggesting that Si3-tail and Si3-body/head can move their positions without restraint. Si3 spans 190 \nthe entire length of cyRNAP, reaching from the secondary channel to the upstream DNA. 191 \nHowever, it likely does not interfere with any basic function of cyRNAP (i.e., DNA binding, 192 \nRNA elongation, binding of initiation factor σ, or elongation factors NusA and NusG), as it runs 193 \nalong the sidewall of cyRNAP (Figs. 2D and 3A). 194 \n During transcription, the secondary channel of all cellular RNAPs, including bacterial 195 \nRNAPs, serves as the only access route between the active site found in the center of RNAP and 196 \nthe external milieu, serving as an entry point for substrate NTPs and an exit route for the RNA 197 \n3’-end during backtracking (prior to RNA cleavage). In cyRNAP, the secondary channel appears 198 \nto be open enough to allow these functions. In addition to these basic functions, the secondary 199 \nchannel serves as a binding platform for proofreading factors such as Gre and regulatory factors 200 \nsuch as DksA, known as secondary channel binding factors (13, 27). These factors use the RNAP 201 \nrim helix as a primary binding site, after which the coiled-coil domain is inserted to access the 202 \nactive site of RNAP (Fig. 3B). In cyRNAP, Si3-tail and -fin occupy the front and bottom sides of 203 \nthe rim helix, respectively, thereby preventing any potential association of secondary channel 204 \nbinding factors (Fig. 3A). 205 \n   206 \nDynamic motion of Si3 associated with the transition between the trigger loop and helix 207 \nduring iNTP binding at the active site 208 \n To investigate the Si3 conformational change associated with trigger helix refolding, we 209 \nprepared an iNTP-bound form of the EC by extending RNA with 3’-deoxy adenosine 210 \ntriphosphate (3’-dATP), which arrested further RNA extension, followed by cytosine 211 \ntriphosphate (CTP) addition as the iNTP (SFig. 6). The resulting cryo-EM structure was 212 \ndetermined at 2.79 Å resolution (SFig. 6). Although an excess amount of CTP was added to the 213 \nEC, a substantial population of ECs (~40%) remained unbound to iNTP. However, the iNTP-214 \nbound EC could be clearly distinguished from the iNTP-free EC during 3D classification of the 215 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n8 \n \ncryo-EM data process due to its unique Si3 orientation relative to the main body of cyRNAP 216 \nassociated with iNTP binding (SFig. 6B and 6D). This allowed for a well-defined density map of 217 \nthe cyRNAP active site. In the iNTP-bound EC, the B-site Mg 2+ (known as the nucleotide-218 \nbinding metal) was present at the active site. However, the A-site Mg 2+ (known as the catalytic 219 \nmetal) was absent, likely due to the lack of a hydroxyl group at the 3’-end of the RNA. Trigger 220 \nhelix folding establishes several essential contacts between the iNTP and amino acid residues, 221 \nincluding β ’2-M339 in contact with the nucleobase and β ’2-H343 in contact with the β-222 \nphosphate group (SFig. 6D). 223 \n Trigger helix folding induces significant motion of Si3 relative to the main body of 224 \ncyRNAP. Specifically, the trigger helix formation pulls a linker connecting the C-terminal half 225 \nof the trigger helix and the Si3-fin, and during this process, the tip of the rim helix acts as a pivot 226 \npoint, converting the lateral motion of the linker (~10 Å) into the rotational motion of Si3, 227 \nresulting in an ~50 Å distance and a 24° swing of Si3-head (Figs. 4A and B, SMovie 2). Si3-228 \nbody/head swings down from the main body of cyRNAP; thus, the β  protrusion domain no 229 \nlonger contacts Si3-body/head in the iNTP-bound EC (Fig. 4A). Remarkably, the large swinging 230 \nof Si3, which is coupled to trigger helix formation (Fig. 4B), did not markedly alter the catalytic 231 \nproperties of cyRNAP (Fig. 4C). Three ECs containing 14, 15 and 16 nucleotide long RNAs 232 \n(EC14, 15 and 16) were prepared by extending the initial 5’-labelled 13 nt long RNA in the 233 \nnucleic acid scaffold shown above the summary table. Nucleotide addition, its direct reversal by 234 \npyrophosphorolysis, and transcript cleavage were performed for the ECs that formed with either 235 \nwild-type (WT) or Si3-lacking ( ΔSi3) cyRNAP. Rates of the NTP addition, pyrophosphorolysis 236 \nand RNA hydrolysis were similar between the WT and ΔSi3 cyRNAPs (Fig. 4C and SFig. 7). 237 \nThe relative rates of these reactions also allowed us to attribute a predominant translocation state 238 \nto the EC tested because nucleotide addition proceeded from post-translocation, 239 \npyrophosphorolysis from pre-translocation and hydrolysis from the backtracked state (scheme on 240 \nFig. 4C). Comparison of the rates of these reactions for the three complexes used in the present 241 \nstudy suggested that EC14 is mainly stabilized in a post-translocated state (characterized by fast 242 \nNTP addition), EC15 is mainly pre-translocated (fast pyrophospholysis), and EC16 is mainly 243 \nbacktracked/paused (faster hydrolysis), similar to the ECs formed by Thermus aquaticus RNAP 244 \n(21), which doesn’t contain Si3, on this template. These results imply that Si3 does not influence 245 \nthe catalysis or translocation equilibrium of cyRNAP. 246 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n9 \n \n The cryo-EM structure of the cyRNAP-promoter DNA complex containing σA (both 247 \nfrom Synechocystis sp. PCC 6803, which is closely related to the S. elongatus PCC 7942 used in 248 \nthis study), promoter DNA and 4-mer RNA was determined by Shen et al. (15); the results 249 \nshowed that Si3-head contacts σA domain 2. This interaction clamps the single-stranded DNA 250 \naround the -10 region, stabilizing the open complex and facilitating transcription initiation. 251 \nComparison of the structures of the cyRNAP promoter complex (15) with those of the EC (this 252 \nstudy) revealed that Si3-body and -head move toward σA domain 2 for interaction but that the 253 \nother cyRNAP structures, including Si3-tail and -fin and the main body of the RNAP, are nearly 254 \nidentical (Fig. 5A). 255 \n Si3 wraps around the main body of cyRNAP, which may facilitate RNAP folding, 256 \nsubunit assembly and/or maturation to form an active and mature form of RNAP as DNA and a 257 \nσ factor that enhances reconstitution of E. coli RNAP (22). To test the function of Si3 during 258 \ncyRNAP assembly and maturation, we performed a refolding experiment with WT, ΔSi3 259 \ncyRNAP and ΔSi3 cyRNAP in combination with the separately expressed and purified Si3 260 \nprotein ( ΔSi3+Si3) (Fig. 5B). The proteins were denatured with 6 M guanidine-HCl and 261 \nrenatured by gradual removal of guanidine-HCl via dialysis against renaturation buffer. The 262 \nactivities of the reconstituted ΔSi3 cyRNAP in the absence and presence of the Si3 protein, as 263 \njudged by their ability to extend 13 nt long RNA in the assembled duplex with template DNA 264 \noligonucleotide, were nearly the same as those of the WT cyRNAP, indicating that Si3 does not 265 \nplay a role in cyRNAP assembly and maturation. This conclusion is supported by the similar 266 \nyields of recombinant WT and ΔSi3 cyRNAPs routinely isolated from E. coli. Remarkably, 267 \nhowever, the separate Si3 protein binds ΔSi3 cyRNAP but not the WT cyRNAP when it is added 268 \nexternally to cyRNAP (Fig. 5C). When complex formation between Si3 and ΔSi3 cyRNAP was 269 \nassessed by a blue native polyacrylamide gel electrophoresis, a band with a lower mobility 270 \nsimilar to that of the WT cyRNAP was observed (Fig. 5C, Lane 4). Interaction between WT 271 \ncyRNAP and Si3 was not detected, i.e., no complex with lower mobility than that of WT 272 \ncyRNAP was detected (Lane 5). 273 \n 274 \nDiscussion 275 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n10 \n \n In this study, we determined the structures of cyRNAP Si3 by X-ray crystallography (Fig. 276 \n1) and of cyRNAP EC-NusG with and without iNTP by cryo-EM (Figs. 2 and 4). We 277 \ninvestigated the function of Si3 by comparing the catalytic activities of WT and ΔSi3 cyRNAPs. 278 \nThe results of structural and biochemical investigations of cyRNAP showed that Si3 is 279 \naccommodated within the cavities of cyRNAP without compromising its basic activities, that it 280 \nshields the site of secondary channel binding proteins, and that it moves within cyRNAP upon 281 \nbinding of iNTP in the active site. Remarkably, a minor structural transition between the trigger 282 \nloop and trigger helix causes a major swinging motion of Si3 (Fig. 4 and SMovie 2). The 283 \npresence of Si3 in the middle of the trigger loop/helix did not affect cyRNAP catalysis under our 284 \nexperimental conditions (Fig. 4C). Because of the large conformational change that occurs 285 \nduring the transcription reaction, changes in cyRNAP activity could be observed when the 286 \nmotion of Si3 is hindered, such as by binding of external factors. Further proteomics for 287 \nsearching factors binding Si3, structural, single-molecule and biochemical studies are required to 288 \nelucidate its role in regulating transcription by cyRNAP, such as by sensing environmental 289 \nsignals (e.g., trafficking of RNAP or transcription-translation coupling) to optimizing cyRNAP 290 \nactivity. Alternatively, the oscillating motion of Si3 might function as a regulatory signal for 291 \ncellular processes. Photosynthetic cyanobacteria synchronize their gene expression patterns with 292 \ndiurnal light cycles (23). Conceivably, the lack of Si3 movement might trigger initiation of 293 \ncyRNAP hibernation through binding to cellular factors or its oligomerization during the night. 294 \nAdditionally, Si3 movement might help RNAP propel through the densely packed cytoplasm of 295 \ncyanobacteria during transcription. 296 \n The primary proofreading mechanism employed by RNAP involves backtracking 297 \nfollowed by hydrolysis of misincorporated nucleotides at the 3’-end of nascent RNA. This 298 \nprocess is significantly enhanced by elongation factors that bind to RNAP secondary channel, 299 \nsuch as Gre in bacteria, TFS in archaea, and TFIIS in eukaryotes (24). However, unlike the 300 \nabsolute majority of living organisms, cyanobacteria lack Gre factor. The intracellular 301 \nconcentration of Mn2+ is two orders of magnitude greater in cyanobacteria than in other bacteria 302 \nto support photosynthesis (16). It is possible that Mn 2+ replaces the catalytic Mg2+ of RNAP and 303 \nthus promotes misincorporation of NTPs (25, 26). Potentially as a compensating mechanism, 304 \ncyRNAP has been shown to possess proficient intrinsic proofreading activity (7, 27). However, 305 \nthis intrinsic activity is still approximately 10 times lower than the Gre-stimulated activity of E. 306 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n11 \n \ncoli RNAP. Gre-like factors either emerged after the split of cyanobacteria from their last 307 \ncommon ancestor with other bacteria or were subsequently lost. The distinctive characteristics of 308 \ncyRNAP—the absence of Gre/DksA factors and the split of the largest subunit may be 309 \nintrinsically linked to Si3 acquisition. The Si3-tail/fin position around the rim helix of RNAP 310 \nprevents association of secondary channel binding proteins, such as GreA and DksA, with 311 \ncyRNAP (Fig. 3). As secondary channel binding proteins play critical roles in transcription 312 \nfidelity and regulation in bacteria, the Si3-GreA/DksA trade-off in cyanobacteria might be 313 \nadvantageous but remains to be fully understood. With Si3 acquisition, β ’ increased to 210 kDa 314 \nin size, and separation of the original rpoC gene into two genes was perhaps beneficial to 315 \nfacilitate expression of such a large protein. The observed change in the position and mobility of 316 \nSi3 in cyRNAP ECs compared to those in the promoter complex (Fig. 5A) raises questions about 317 \nthe role of Si3 in promoter escape. Si3 may complicate promoter escape by binding to the σ 318 \nfactor; conversely, its large-range movement upon RNA synthesis may contribute to weakening 319 \nσ association with core and/or promote σ release at transition to elongation stage. 320 \n The structure corresponding to Si3 of cyRNAP has not been found in other bacterial 321 \nRNAPs. However, the structure and arrangement of the Rpb9 subunit in eukaryotic RNAPII 322 \nshow remarkable similarity to those of the Si3 subunit of cyRNAP (Fig. 3C). Rpb9 is positioned 323 \nwithin a cavity between the rim helix and the lobe domain of RNAPII, akin to the Si3-fin of 324 \ncyRNAP (highlighted in red in cyRNAP and RNAPII). Rpb9 is a unique subunit found only in 325 \nRNAPII and plays a critical role in enhancing the accuracy of transcription (28). Although both 326 \nRpb9 and Si3-tail are located away from the active site of RNAP, their presence may enhance 327 \ntranscription fidelity, which coordinates RNAP confirmation changes such as RNAP swiveling 328 \nand/or movement of the rim helix during the nucleotide addition cycle (20). The presence of 329 \nthese unique structural features in different types of RNAPs suggests a common mechanism for 330 \nenhancing transcriptional accuracy and specificity across different organisms. Further 331 \ninvestigation of Si3 function at different stages of transcription and under several growth 332 \nconditions in cyanobacteria will be required to determine the full array of its biological 333 \nfunctions. 334 \n 335 \nExperimental Procedures 336 \nProtein preparation 337 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n12 \n \nThe DNA fragment encoding Thermosynechococcus elongatus  BP-1 Si3 in the β’2 subunit 338 \n(TelSi3, RpoC2 residues 345-983, 69 kDa) was cloned between the NdeI and BamHI sites of the 339 \npET15b expression vector to introduce an N-terminal His 6-tag, and the protein was 340 \noverexpressed in E. coli BL21(DE3)/pLysS cells. Transformants were subsequently grown in LB 341 \nmedia supplemented with ampicillin (100 μ g/ml) and chloramphenicol (25 μg/ml) at 37 °C until 342 \nthe OD600 reached ~0.5, after which protein expression was induced by adding 0.5 mM IPTG for 343 \n10 h at 4 °C. The harvested cells were lysed by sonication, and proteins in the soluble fraction 344 \nwere purified by Ni-affinity column chromatography (HisTrap 5 ml column, GE Healthcare). 345 \nThe His6-tag was removed by thrombin digestion (1 μg of thrombin per mg of TelSi3 protein) 346 \nfor 20 h at 4 °C, and the protein was further purified by Q Sepharose column chromatography 347 \n(GE Healthcare) and gel-filtration column chromatography (HiLoad Superdex75 16/60, GE 348 \nHealthcare). The purified protein was concentrated to 15 mg/ml and exchanged into buffer 349 \ncontaining 10 mM Tris-HCl (pH 8.0), 50 mM NaCl and 0.1 mM EDTA. 350 \n 351 \nLimited trypsinolysis 352 \nLimited trypsinolysis was used to remove flexible regions from TelSi3, and N-terminal amino 353 \nacid sequencing was used to identify protein fragments suitable for crystallization. The trypsin 354 \ndigests were carried out in 10 mM Tris–HCl (pH 8), 100 mM NaCl, 5% (v/v) glycerol, 0.1 mM 355 \nEDTA and 1 mM DTT. TelSi3 (10 mg/ml) was digested in a 10 µl volume with different 356 \namounts of trypsin (5 nM to 5 µM) for 10 min at 25 °C. The reactions were terminated by 357 \naddition of PMSF. The trypsinized fragments were separated by SDS /i2 PAGE and blotted onto 358 \nPVDF membranes, and the N-terminal sequences were determined by Edman based protein 359 \nsequencing. The TelSi3 fragment containing residues 435-938 (TelSi3ΔN, 60 kDa) was PCR 360 \nsubcloned and inserted into the pET15b expression vector between the NdeI and BamHI sites. 361 \nThe protein was overexpressed and purified as described above for full-length TelSi3. 362 \n 363 \nCrystallization 364 \nInitial crystals of TelSi3 ΔN were obtained by the hanging-drop vapor diffusion method by 365 \nmixing equal volumes of the protein solution (20 mg/ml) and crystallization solution (0.1 M 366 \nsodium citrate [pH 3.5], 0.2 M MgCl 2 and 10% PEG6000) and incubating at 4 °C over the same 367 \ncrystallization solution. The large crystals (0.5 × 0.2 × 0.2 mm) used for X-ray data collection 368 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n13 \n \nwere prepared by microseeding by mixing 2 µl of protein solution, 2 µl of crystallization solution 369 \n(0.1 M sodium citrate [pH 5], 0.4~0.6 M MgCl 2, 4~6% PEG3350 and 50 µg/ml heparin) and 0.2 370 \nµl of seed solution. The crystals were then dehydrated by transfer to crystallization solution 371 \n(without heparin) with increasing concentrations of PEG3350 (in 5% steps) to a final 372 \nconcentration of 20% and incubated for 5-10 h. For all procedures, crystal preparation, growth 373 \nand dehydration were performed at 4 °C. The crystals were transferred to a crystallization 374 \nsolution with 25% (v/v) propylene glycol as a cryoprotective solution and flash frozen in liquid 375 \nnitrogen. Selenomethionine-substituted proteins were prepared for SAD analysis by suppressing 376 \nmethionine biosynthesis. 377 \n 378 \nX-ray data collection and crystal structure determination 379 \nIn addition to the four original methionine residues found in TelSi3 ΔN (including an N-terminal 380 \nmethionine residue resulting from cloning into the pET15b vector), three methionine residues 381 \nwere introduced by replacing the leucine residues at 508, 738 and 922 by site-directed 382 \nmutagenesis to obtain the experimental phase via single-anomalous dispersion (SAD) 383 \nexperiments using SeMet-labeled proteins. The TelSi3ΔN protein with seven selenomethionine 384 \nresidues (TelSi3 ΔNMet7) was generated by suppressing methionine biosynthesis during 385 \noverexpression of the TelSi3ΔNMet7 protein. The protein was purified as described above. 386 \nDiffraction data were corrected at National Synchrotron Light Source (NSLS) beamline X25. 387 \nThere are six TelSi3 ΔNMet7 molecules in an asymmetric unit of the crystal belonging to the 388 \nP3(2)21 space group. The crystallographic datasets were processed using HKL2000 (29). With 389 \nthe anomalous signal from SeMet, the experimental phase (figure of merit: 0.273) was calculated 390 \nusing automated structure solution (AutoSol) in PHENIX (30). Density modification yielded a 391 \nmap suitable for manual model building by Coot (31) followed by structure refinement using 392 \nPHENIX. The final coordinates and structure factors have been deposited in Protein Data Bank 393 \n(PDB) under the accession codes listed in Supplementary Table 1. 394 \n 395 \nExpression and isolation of the Synechococcus elongatus RNAP  396 \nThe core enzyme of cyRNAP was overexpressed in E. coli  T7Express cells (New England 397 \nBiolabs) cells transformed with a pET28a expression vector containing the α , β , β ’1, β ’2 and ω  398 \nencoding genes ( β  and β ’2 contain a Strep-tag and His-tag, respectively) (32).  The cells were 399 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n14 \n \ngrown in LB media supplemented with kanamycin (50 μ g/ml) at 37 °C until the OD600 was ~0.6. 400 \nAfterward, the cells were induced with IPTG (1 mM) and grown overnight at 22 °C. 401 \nThe biomass was harvested and suspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 250 mM 402 \nNaCl, 10% glycerol, 20 mM imidazole, and 1 mM β -mercaptoethanol and protease inhibitors 403 \nfrom Roche according to the manufacturer’s instructions). The cells were sonicated, lysate 404 \ncentrifuged at 18 k x g, after which the supernatant was collected. The protein was purified at 405 \n4 °C sequentially through a HisTrap (5 mL) column and a Strep-Tactin XT (1 mL) column (both 406 \nfrom Cytiva). The latter column was washed with 3 column volumes (CVs) of Buffer W (100 407 \nmM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA). The bound protein was eluted by applying 408 \n1 CV of Buffer E (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM 409 \ndesthiobiotin). The purified cyRNAP (20 μ M) was assessed using SDS/i2 PAGE, dialyzed against 410 \nStorage Buffer (40 mM Tris–HCl pH 8.0, 200 mM KCl, 1 mM EDTA, 1 mM DTT, and 5% 411 \nglycerol), and stored at -80 °C. 412 \n 413 \nCloning, expression and isolation of the Synechococcus elongatus NusG and Si3 proteins 414 \nThe NusG was overexpressed in E. coli  T7Express cells (New England Biolabs) cells 415 \ntransformed with pET28a expression vector where the gene for the C-terminal His 6-416 \ntagged Synechococcus elongatus  NusG was cloned. Cells were grown in LB 417 \nmedium supplemented with kanamycin (50 μ g/ml) at 37°C until OD 600 ~0.5, then induced with 418 \nIPTG (1 mM) and grown overnight at 22°C. Culture pellets were sonicated in 50 ml Lysis Buffer 419 \n(10 mM Tris-HCl pH 7.9, 300 mM KCl, protease inhibitors from Roche according 420 \nto manufacturer), spun at 18K rpm, and filtered through 0.22 μ M syringe filter. Filtered 421 \nsupernatant was subjected to Ni-NTA affinity chromatography in 10 mM Tris-HCl pH 7.9, 600 422 \nmM KCl, 5% glycerol with 50 mM imidazole washes and 100 mM imidazole elution. The eluted 423 \nprotein (in 600 mM KCl) was diluted (~100 mM KCl) and applied to a pre-equilibrated with 424 \n(10 mM Tris-HCl pH 8.0, 100 mM KCl, 5% glycerol) 5 ml Resource Q column, Cytiva. The 425 \ncolumn was washed with 5 CV of equilibration buffer, and the protein was eluted by applying a 426 \nlinear salt gradient (100-1 M KCl) over 10 CV. The purified NusG (90 μ M) was checked using 427 \nSDS-PAGE, stored in Storage Buffer (40 mM Tris-HCl pH 8.0, 200 mM KCl, 1 mM EDTA, 1 428 \nmM DTT, 5% glycerol) at -80 °C. Cyanobacteria-specific loop of NusG (residues 110-122) was 429 \ndeleted by site-directed mutageneses, and the mutant NusG was isolated as the WT protein. 430 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n15 \n \nThe open reading frame encoding separate full-size Si3 domain was cloned into pET28 vector, 431 \noverexpressed  E. coli  T7Express cells (New England Biolabs) as the N-terminal His 6-tagged  432 \nprotein, and isolated via Ni-NTA affinity chromatography on HisTrap column, Cytiva, similarly 433 \nto NusG protein. After affinity chromatography the protein was dyalised against the storage 434 \nbuffer (20 Tris-HCl pH 8.0, 200 mM KCl, 1 mM EDTA, 1 mM DTT, 50% glycerol). 435 \n 436 \nSample preparation for cryo-EM 437 \nThe cyRNAP EC with NusG was reconstituted in vitro by mixing 5 μ M cyRNAP with equimolar 438 \namounts of template DNA and RNA (Fig. 2A) in storage buffer at 37 °C for 10 minutes, 439 \nfollowed by mixing with 7 μ M nontemplate DNA and incubating further for 10 minutes. The 440 \nresulting EC was mixed with 7 μ M NusG and incubated for 10 min at 37 °C. CHAPSO (8 mM) 441 \nwas added to the sample just before vitrification. The iNTP-bound EC was prepared by adding 1 442 \nmM 3’-deoxyATP or CTP to the EC with NusG and incubating for 5 min at 37 °C. Another 443 \ndifference between the EC- and iNTP-bound ECs is the nontemplate DNA used in the scaffold, 444 \nthe latter of which contains complementary transcription bubbles. 445 \n 446 \nGrid preparation for cryo-EM 447 \nC-flat Cu grids (CF-1.2/1.3 400 mesh, Protochips, Morrisville, NC) were glow-discharged for 40 448 \nseconds using the PELCO easiGlowTM system prior to application of 3.5 μ l of the sample (2.5 –449 \n3.0 mg/ml protein concentration) and plunge-freezing in liquid ethane using a Vitrobot Mark IV 450 \n(FEI, Hillsboro, OR) with 100% chamber humidity at 5 °C. 451 \n 452 \nCryo-EM data acquisition and processing 453 \nData were collected using a Titan Krios (Thermo Fisher) microscope equipped with a Falcon IV 454 \ndirect electron detector (Gatan) at Penn State Cryo-EM Facility. Sample grids were imaged at 455 \n300 kV, with an intended defocus range of -2.5 to -0.75 μ m and a magnification of 75,000X in 456 \nelectron counting mode (0.87 Å per pixel). Movies were collected with a total dose of 45 457 \nelectrons/Å2. Downstream processing was performed with CryoSPARC (33). The movies were 458 \ncorrected and aligned using patch motion correction followed by patch CTF correction. Particles 459 \nwere picked using a template-based autopicker and multiple rounds of 2D classification to 460 \ndiscard bad particles. The 2D classes with EC-NusG particles were selected and used for training 461 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n16 \n \nthe Topaz model (34). The Topaz-extracted particles were subjected to multiple rounds of 462 \nheterogeneous refinement to remove junk particles. Finally, a nonuniform refinement operation 463 \nwas run on the final set of particles to yield the reconstruction (SFigs. 4 and 6). 464 \n 465 \nStructure refinement and model building 466 \nA model of the cyRNAP core enzyme was constructed by homology modeling using core RNAP 467 \nfrom the cryo-EM structure of the Syn6803 RNAP- σA promoter DNA open complex as a 468 \nreference model. A model of cyNusG was constructed with the AlphaFold2 gene (35). DNA and 469 \nRNA models were constructed using the E. coli RNAP elongation complex (PDB: 7MKO) as a 470 \nguide. The cyRNAP gene was manually fitted into the cryo-EM density map using Chimera (36), 471 \nfollowed by rigid body and real-space refinement using Coot (31) and Phenix (37). 472 \n 473 \nIn vitro transcription in the assembled elongation complexes 474 \nECs were assembled and immobilized as described (38). Sequences of the oligonucleotides used 475 \nfor the assembly of ECs are shown on Fig.4C. For assembly of ECs used for experiments on Fig. 476 \n4C, 13 nt long RNA was radiolabelled at the 5’-end with [ γ -32P] ATP and T4 Polynucleotide 477 \nkinase (New England Biolabs) prior to complexes assembly.  Stalled elongation complexes 478 \nEC14, EC15 and EC16 were obtained by extension of the initial RNA13 in EC13 with 10 μ M 479 \nNTP sets according to the sequence for 5 min and then were washed with TB to remove Mg 2+ 480 \nand NTPs. Reactions were initiated by addition of 10 mM MgCl 2 with or without either 1 μ M 481 \nNTPs or 250 μ M PPi. Single nucleotide addition and pyrophosphorolysis experiments were 482 \nperformed at 30°C in transcription buffer (TB) containing 20 mM Tris–HCl pH 6.8, 40 mM KCl, 483 \n10 mM MgCl 2, transcript hydrolysis was done in the same buffer except at pH 7.9. After 484 \nincubation for intervals of time specified on Figures, reactions were stopped with formamide-485 \ncontaining buffer. Products were resolved by denaturing 23% polyacrylamide gel electrophoresis 486 \n(PAGE) (8 M Urea), revealed by PhosphorImaging (Cytiva) and visualized using ImageQuant 487 \n(Cytiva) software. Kinetics data were fitted to a single exponential equation y=y 0+a-bx using 488 \nSigmaPlot software by non-linear regression to determine rate constants of the reactions.  489 \n 490 \nDenaturation and renaturation of cyRNAP 491 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n17 \n \nDenaturation of cyRNAP was performed by incubating the purified protein for 20 min in 492 \ndenaturing buffer containing 20 mM Tris-HCl (pH 7.9), 6 M guanidine-HCl, 5% glycerol, 1 mM 493 \nEDTA, and 10 mM DTT at 30 °C in a 100 µl volume and with a cyRNAP concentration of 0.5 494 \nmg/ml. Recombinant Si3 was included in 2.5 molar excess. The proteins were renatured via 495 \novernight dialysis at 7 °C against renaturing buffer containing 20 mM Tris-HCl (pH 7.9), 200 496 \nmM KCl, 10% glycerol, 2 mM MgCl2, 10 µM ZnCl2, 1 mM EDTA, and 1 mM DTT. Aliquots of 497 \nthe renaturation mixture and their serial dilutions were used for nucleotide addition experiments 498 \non assembled constructs containing template DNA and RNA oligonucleotides. A 13 nt RNA 499 \noligonucleotide was radiolabeled at the 5’ end with [ γ -32P] ATP and T4 polynucleotide kinase 500 \n(New England Biolabs) prior to EC assembly. The indicated on the Fig. 5C amount of assembly 501 \n# 502 \nmixture was incubated with the RNA-DNA duplex for 5 min at room temperature, then 10 µM 503 \nGTP was added for 10 minutes at 30°C. Reactions were stopped and products analyzed as 504 \nbefore. 505 \n 506 \nComplex formation between the Si3 protein and core cyRNAP 507 \nFor the binding experiment 150 nM core enzymes and 1.5 µM Si3 proteins were incubated for 10 508 \nminutes at 4°C in 20 mM Tris-HCl pH 7.9, 40 mM KCl, mixed with loading dye (final 509 \nconcentration is 50mM BisTris pH 7.2, 50mM NaCl, 10% glycerol, 0.001% Ponceau S) and 510 \nresolved on the NativePAGE 3-12% Bis-Tris gel, Invitrogen using running buffers prepared 511 \naccording to the manufacturer, for 90 minutes at 150V. Gel was fixed with 50% methanol,10% 512 \nacetic acid solution, and additionally de-stained by boiling in 8% acetic acid.   513 \n 514 \nSalt stability of elongation complexes 515 \nElongation complex was assembled using oligos shown on SFig. 5B. 14 nt RNA in ECs on was 516 \nradiolabelled at the 3’ end by incorporation of [ α -32P] GTP into original 13 nt long RNA. To 517 \nexamine the stability of ECs, ECs bound to the streptavidin sepharose beads, Cytiva via strep tag 518 \non β  subunit of cyRNAP, were incubated in TB containing 300 mM KCl at 30°C for times 519 \nspecified on SFig. 5B. WT or mutant NusG Δ110-122 were added where specified at 1 μ M final 520 \nconcentration. Supernatant and total fractions were collected for analysis. Reactions were 521 \nstopped and products analyzed as before. 522 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n18 \n \n 523 \nFigure legends 524 \nFig. 1. X-ray crystal structure of TelSi3Δ N. (A) The thick bars represent the primary sequences 525 \nof the largest subunits of the bacterial, chloroplast and archaeal RNAPs. Domains (Si3, green 526 \nboxes) and structural motifs (RH, rim helix; BH, bridge helix; TL, trigger loop) are labeled. The 527 \nlettered boxes represent evolutionarily conserved regions. The split ends of the two polypeptides 528 \nare indicated by black triangles. (B) Crystals of TelSi3 ΔN. (C) Structure of TelSi3 ΔN. Six 529 \nmolecules of TelSi3 ΔN (I~VI) are present in the asymmetric unit. Molecules are depicted as 530 \ncartoon models with transparent surfaces, and each molecule is denoted by a unique color and 531 \nlabeled. (D) The backbone is colored as a ramp from the N-terminus to the C-terminus, from 532 \nblue/cyan/green/yellow/orange/red. SBHMs are labeled 1 to 8, and subdomains (tail, fin, body 533 \nand head) are indicated. The TelSi3 ΔN structure lacks SBHM-1, and the trigger loops (TL N and 534 \nTLC) are depicted as blue oval and pink cylinders, respectively, with black lines showing their 535 \nconnections with TelSi3 ΔN. (E) Molecules 1 and 3 of TelSi3 ΔN are superimposed via fin 536 \nsubdomains, revealing flexibility in the orientation between the fin and body/head subdomains. 537 \n 538 \nFig. 2. Cryo-EM image of the cyRNAP elongation complex with NusG.  (A) The sequence of 539 \nthe DNA/RNA scaffold used for the EC-NusG assembly (template DNA, green; nontemplate 540 \nDNA, yellow; RNA, red). DNA and RNA regions lacking cryo-EM density are underlined. (B) 541 \nOrthogonal views of the cryo-EM density map. Subunits and domains of cyRNAP, DNA, RNA 542 \nand NusG are colored and labeled (RH, rim helix; prot, protrusion; downDNA, downstream 543 \nDNA; upDNA, upstream DNA). The split ends of the β ’1 and β ’2 subunits are indicated by 544 \nwhite circles. The SBHMs in Si3 are labeled 1 to 8. (C) Cryo-EM density of DNA, RNA and 545 \nNusG are shown with a transparent RNAP density map (ntDNA, nontemplate DNA; ssRNA, 546 \nsingle-stranded RNA). The 5’ and 3’ ends of the RNA are indicated. The cryo-EM density map 547 \nis colored according to B. (D) Efficient storage of an elongated and large Si3 molecule on the 548 \nsurface of cyRNAP. The structure of EC-NusG is shown as a transparent surface, and the Si3, 549 \nDNA/RNA and trigger loop (TL N, TL C) regions are shown as cartoon models. SBHMs are 550 \nlabeled 1 to 8, and subdomains (tail, fin, body and head) are indicated. The active site of RNAP 551 \nis designated by catalytic Mg2+ (magenta sphere). 552 \n 553 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n19 \n \nFig. 3. Comparison of the structures of cyRNAP, E. coli RNAP and eukaryotic RNAPII. 554 \nThe structures of cyRNAP ( A), E. coli RNAP with GreB (PDB: 6RIN, B) and yeast RNAPII 555 \n(PDB: 7ML0, C) are shown as transparent surfaces with domains, subunits and a factor 556 \ndescribed in the main text. 557 \n 558 \nFig. 4. Si3 movement during the trigger helix folding. (A) Cryo-EM maps of the iNTP-bound 559 \n(gray) and iNTP-free (light blue) states of the EC-NusG strains (RH, rim helix; prot, protrusion; 560 \nupDNA, upstream DNA). Arrows indicate movement of Si3 by trigger helix folding. (B) 561 \nConformational change in Si3 during the transition from the trigger loop (TL) to the trigger helix 562 \n(TH) by iNTP (blue stick model) binding. The red and black arrows indicate movements of the 563 \nTL/TH-Si3 linker and Si3, respectively. A pivot point for converting the movement of the linker 564 \nto the swing motion of Si3 is shown as a blue transparent circle. (C) Si3 does not influence 565 \ncatalysis by cyRNAP. Scheme and sequence of the assembled elongation complex used for 566 \nexperiments with WT and ΔSi3 RNAPs. The table represents the summary of reaction rate 567 \nconstants of single nucleotide addition (kNTP), pyrophosphorolysis (kPPi) and transcript hydrolysis 568 \n(kOH-) in EC14, EC15 and EC16 by WT and ΔSi3 RNAPs. The values that follow the ± sign are 569 \nthe values of standard deviation derived from three independent experiments. The shade of green 570 \nin the cells reflects the value of the constant, i.e., darkest shade corresponds to the highest rate. 571 \nThe right column shows the predominant translocation states of the elongation complexes, as 572 \ndeduced from the relative rates of reaction. Scheme of RNAP oscillation in translocation 573 \nequilibrium and the architecture of the nucleic acid scaffold of the elongation complex in 574 \npost/i2 translocation, pre /i2 translocation and backtracked states, as adapted from (21). The 575 \ntemplate DNA, the non ‐ template DNA and the RNA are green, yellow and pink, respectively. 576 \nCatalytic Mg2+ ions and the i+1 site of the RNAP active center are shown by a red circle and a 577 \nblue rectangle, respectively.  578 \n 579 \nFig. 5. Si3 functions. (A) The cryo-EM structures of cyRNAP in the EC (left) and the promoter 580 \ncomplex (right, PDB: 8GZG). The contact between Si3-head and σA in the promoter complex is 581 \nindicated by a black circle. (B) Si3 is not required for cyRNAP assembly or maturation. WT and 582 \nΔSi3 cyRNAPs were denatured and subsequently renatured, after which their activity was tested 583 \non the construct mimicking the DNA template–RNA transcript duplex structure by their ability 584 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n20 \n \nto incorporate the next nucleotide, G, dictated by the template. Twofold serial dilutions of the 585 \nassembly mixture with the indicated initial amounts of core enzymes were tested. The vertical 586 \nlines indicate the positions where the parts of the same gel were combined. (C) The recombinant 587 \nSi3 protein can bind ΔSi3 cyRNAP but not WT cyRNAP. The complex formation between the 588 \nindicated proteins was analyzed by blue native polyacrylamide gel electrophoresis. The vertical 589 \nline indicates the position where two parts of the same gel were combined. 590 \n 591 \nData, Materials, and Software Availability.  The X-ray crystallographic density map and the 592 \nrefined model have been deposited in Protein Data Bank (www.rcsb.org) under accession 593 \nnumber 8EMB. The cryo-EM density map and the refined model have been deposited in 594 \nElectron Microscopy Data Bank (www.ebi.ac.uk/emdb/) under accession numbers EMD-40874 595 \n(iNTP-free EC-NusG) and EMD-42502 (iNTP-bound EC-NusG) and in Protein Data Bank 596 \n(www.rcsb.org) under accession numbers 8SYI (iNTP-free EC-NusG) and 8URW (iNTP-bound 597 \nEC-NusG). All study data are included in the article and/or SI Appendix. 598 \n 599 \nACKNOWLEDGMENTS 600 \nWe thank Jean-Paul Armache at Penn State for the technical support. We thank the National 601 \nSynchrotron Light Source (NSLS) Brookhaven National Laboratory for X-ray data collection.  602 \nWe would like to acknowledge the Penn State Huck Life Science Institutes Cryo-EM Core 603 \nFacility for use of the Talos Arctica G2 TEM and the Vitrobot Mark IV and Sung Hyun Cho for 604 \ndata collection. We thank Yu Zhang at the Shanghai Institute of Plant Physiology and Ecology 605 \nfor kindly sharing the coordinates of Synechocystis sp. PCC 6803 RNAP. This work was 606 \nsupported by a National Institutes of Health grant (R35 GM131860 to K. S. M.) and a 607 \nBiotechnology and Biological Sciences Research Council grant BB/W017385/1 to Y.Y. 608 \n 609 \nREFERENCES 610 \n1. T. Borner, A. Y. Aleynikova, Y. O. Zubo, V. V. Kusnetsov, Chloroplast RNA 611 \npolymerases: Role in chloroplast biogenesis. Biochim Biophys Acta 1847, 761-769 612 \n(2015). 613 \n2. T. 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It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\nheadbodyfin\n24 Å, 11º\nI\nII\nIII V\n8\n2\n3 4\n5\n6\n7\nTLc\nTLN\n1\nMg (active site)\n0.5 x 0.2 x 0.2 (mm)\nC\nB D\nE\ntail\nFig. 1 \nA\nA\nA\nC\nC\nC\nB\nB\nB\nD\nD\nD\nF\nF\nF\nE\nE\nE\nG\nG\nG\nH\nH\nH\n645\n793\n188\nG\nG\nGE. coli\nCyanobacteria\n(T. elongatus)\n(S. elongatus)\nChloroplast\n(A. thaliana)\nBH\nRH\nTLN TLC JawMg2+\nA CB D FE G HArchaeal RNAP\nTL\nA\nclump\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\nSi3\nupDNAb’1\nb’2\na\nRNA ch\n1\n8\nprot\nlobe\nRH\n３ ４ 5\n6\n7\nb\nRNA ch\nflap\nupDNA\nNusGSi3\nb\nw\na\nb’1\nprot\n1 8\nRH\nw\nsplit \nends \na\n2nd ch\n82\nb\nb’2\n lobe\n2\n3 4\n5\n6\n7\n8\nTLc\nTLN\nMg\nRNAdownDNA\nfin body\nheadtail 8\n1\nupDNA\nB\nA  CCTCTCCATG\n5'-GGGCGCATGCTGCTCTA ACGGCGACTGCCC-3’\n3'-CCCGCGTACGACGAGATCCTCTCCATGTGCCGCTGACGGG-5’\n                    GGAGAGGUA\n      5'-GCAUUCAAAGC\nupDNA downDNA\nRNA\nC\ndownDNA\nupDNA\nNusG\n3’\n5’\nntDNA\nssRNA\nFig. 2 \nD\n2nd ch\n90°\ndownDNA\nSi3\nb’1\nb’2\nNusG\n1\n2\n8\nlobe\nclamp\njaw\n３ ４\n5\n6\n7\n90° 180°\nMg\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\n90°\nSi3-body/head\nMg\nBH\nRH\nSi3-tail\nSi3-fin lobe\nSi3-fin\nRH\nBH\nMg\nSi3-tail\nlobe\n2nd ch\nSi3-body/head\nMg\nBH\nRH\nGreB\nlobe\nSi3\nMg\nBH\nRH\nGreB\nlobeSi3\nMg\nBH\nRH\nlobe\nRpb9\nMg\nBH\nRH\nlobe\nRpb9\ncyRNAP\nE. coli RNAP-GreB\nYeast RNAPII\nacidic residues\nFig. 3 \nA\nB\nC\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\nprot\nlobe\nhead\nRH\nbody\nfin\ntail\nupDNA\n RH\nfin\ntail\n2nd ch\nhead\nbody\n50 Å, 24º\nA\nfin\ntail\nTLc\nTLN\nMgA\nBH\ndownDNA\nTHc\nTHN\nNusG\niNTP\nMgB\ntDNA\nntDNA\nRNA\nbody\n head\nupDNA\nSi3\niNTP\nFig. 4 \n90°\nB RH\nfin\ntail\nbody\nhead\nC\npivot point\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint \n\nupDNA\nb\na\nNusG\nSi3\npDNA\nb’1\nb’2\nsA\n-35\n-10\nheadbody\ntail\nfin\ncyRNAP-sA  holoenzyme \npromoter DNA complex\ncyRNAP EC with NusG\nA\nFig. 5 \nB\nC\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575193doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}