The impact of leadered and leaderless gene structures on translation efficiency, transcript stability, and predicted transcription rates inMycobacterium smegmatis

ABSTRACT Regulation of gene expression is critical for the pathogen Mycobacterium tuberculosis to tolerate stressors encountered during infection, and for non-pathogenic mycobacteria such as Mycobacterium smegmatis to survive stressors encountered in the environment. Unlike better studied models, mycobacteria express ∼14% of their genes as leaderless transcripts. However, the impacts of leaderless transcript structures on mRNA half-life and translation efficiency in mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5’ UTRs to mRNA half-life and translation efficiency are similarly unknown. In both M. tuberculosis and M. smegmatis , the essential sigma factor, SigA, is encoded by an unstable transcript with a relatively short half-life. We hypothesized that sigA ’s long 5’ UTR caused this instability. To test this, we constructed fluorescence reporters and then measured protein abundance, mRNA abundance, and mRNA half-life. From these data we also calculated relative transcription rates. We found that the sigA 5’ UTR confers an increased transcription rate, a shorter mRNA half-life, and a decreased translation rate compared to a synthetic 5’ UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts produced less protein compared to any of the leadered transcripts. However, translation rates were similar to those of transcripts with the sigA 5’ UTR, and the protein levels were instead explained by lower transcript abundance. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein:mRNA ratios for natural leadered and leaderless transcripts, consistent with the idea that variability in translation efficiency among mycobacterial genes is largely driven by factors other than leader status. The variability in mRNA half-life and predicted transcription rate among our constructs could not be explained by their different translation efficiencies, indicating that other factors are responsible for these properties and highlighting the myriad and complex roles played by 5’ UTRs and other sequences downstream of transcription start sites.