The Impact of Leadered and Leaderless Gene Structures on Translation Efficiency, Transcript Stability, and Predicted Transcription Rates in Mycobacterium smegmatis

Abstract

Regulation of gene expression is critical for Mycobacterium tuberculosis to tolerate stressors encountered during infection and for nonpathogenic mycobacteria such as Mycobacterium smegmatis to survive environmental stressors. 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′ untranslated regions (UTRs) to mRNA half-life and translation efficiency are similarly unknown. In M. tuberculosis and M. smegmatis, the essential sigma factor, SigA, is encoded by a transcript with a relatively short half-life. We hypothesized that the long 5′ UTR of sigA causes this instability. To test this, we constructed fluorescence reporters and measured protein abundance, mRNA abundance, and mRNA half-life and calculated relative transcript production rates. The sigA 5′ UTR conferred an increased transcript production rate, shorter mRNA half-life, and decreased apparent translation rate compared to a synthetic 5′ UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts appeared to be translated with similar efficiency as those with the sigA 5′ UTR but had lower predicted transcript production rates. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein/mRNA ratios for leadered and leaderless transcripts, suggesting that variability in translation efficiency is largely driven by factors other than leader status. Our data are also discussed in light of an alternative model that leads to different conclusions and suggests leaderless transcripts may indeed be translated less efficiently. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, is a major public health problem killing 1.5 million people globally each year. During infection, M. tuberculosis must alter its gene expression patterns to adapt to the stress conditions it encounters. Understanding how M. tuberculosis regulates gene expression may provide clues for ways to interfere with the bacterium’s survival. Gene expression encompasses transcription, mRNA degradation, and translation. Here, we used Mycobacterium smegmatis as a model organism to study how 5′ untranslated regions affect these three facets of gene expression in multiple ways. We furthermore provide insight into the expression of leaderless mRNAs, which lack 5′ untranslated regions and are unusually prevalent in mycobacteria.

Regulation of mRNA Stability During Bacterial Stress Responses

“…Most studies measure half-life by measuring decreases in mRNA abundance following transcription blockage by rifampicin. Variability may arise from the time-points chosen to assay abundance following transcriptional block, given that we and others have reported multiphasic decay kinetics (Hambraeus et al, 2003;Selinger et al, 2003;Chen et al, 2015;Nguyen et al, 2020). Methodology for normalization and for calculating half-lives also vary (see Table 1).…”

“…Studies of various mRNAs have identified multiple features that confer protection against RNase cleavage ( Figures 3 , 4A ). These include stem-loops ( Emory et al, 1992 ; McDowall et al, 1995 ; Arnold et al, 1998 ; Hambraeus et al, 2002 ), 5′ UTRs and leader/leaderless status ( Chen et al, 1991 ; Arnold et al, 1998 ; Unniraman et al, 2002 ; Nguyen et al, 2020 ), subcellular compartmentalization ( Khemici et al, 2008 ; Montero Llopis et al, 2010 ; Murashko et al, 2012 ; Khemici et al, 2015 ; Moffitt et al, 2016 ), 5′ triphosphate groups ( Bouvet and Belasco, 1992 ; Emory et al, 1992 ; Arnold et al, 1998 ; Mackie, 1998 ), 5′ NAD + /NADH/dephospho-coenzyme A caps ( Chen et al, 2009 ; Kowtoniuk et al, 2009 ; Bird et al, 2016 ; Frindert et al, 2018 ), Np n N caps ( Luciano et al, 2019 ; Hudecek et al, 2020 ), and association with regulatory proteins and sRNAs ( Braun et al, 1998 ; Gualerzi et al, 2003 ; Moll et al, 2003 ; Afonyushkin et al, 2005 ; Daou-Chabo et al, 2009 ; Nielsen et al, 2010 ; Morita and Aiba, 2011 ; Faner and Feig, 2013 ; Liang and Deutscher, 2013 ; Deng et al, 2014 ; Sinha et al, 2018 ; Zhao et al, 2018 ; Cameron et al, 2019 ; Chen H. et al, 2019 ; Richards and Belasco, 2019 ). For example, in Streptococcus pyogenes the sRNA FasX binds to the 5′ end of ska – a transcript coding for streptokinase – increasing its mRNA half-life, thus allowing an extended period of time in which translation of streptokinase can occur ( Ramirez-Pena et al, 2010 ).…”

Translation of a leaderless reporter is robust during exponential growth and well sustained during stress conditions in Mycobacterium tuberculosis

“…Previous research in M. tuberculosis has shown that approximately 25% of its genes are expressed as leaderless transcripts 15,16 , a substantially higher percentage than that reported in other bacterial pathogens (1.2-3%) [17][18][19][20] . In the model organism Mycobacterium smegmatis, which also contains a similar percentage of leaderless transcripts to that of M. tuberculosis, comparable translation rates for canonical and leaderless transcripts have been reported 16,21 . Altogether, this suggests that leaderless translation might have a more central role in the regulation of mycobacterial physiology than in that of E. coli.…”

“…To identify possible differences in the translation e ciencies of the Shine-Dalgarno and leaderless reporters that could be associated with the growth status of the bacteria we used pTC2 and pTC5 to closely monitor luminescence production during the switch from exponential to stationary growth. As a control, we also measured translation of the reporters in M. smegmatis as experiments using uorescent reporters have shown that leaderless translation in M. smegmatis, a close relative of M. tuberculosis that also encodes a high percentage of leaderless proteins in its genome 15,16 , has similar e ciency to that of a Shine-Dalgarno reporter 21 . Additionally, we measured translation of the reporters in E. coli, a bacterium with scarce leaderless transcripts that are translated at low levels by the 70S monosome 25 .…”

Chemotranscriptomic Profiling Defines Drug-Specific Signatures of the Glycopeptide Antibiotics Dalbavancin, Vancomycin and Chlorobiphenyl-Vancomycin in a VanB-Type-Resistant Streptomycete

“…Absence of a 5′-UTR leader sequence can impact the rate of transcript synthesis and degradation, through altered mRNA stability and reduced opportunities for interaction with regulatory sRNA molecules and also the rate of its translation, through a requirement for a different mechanism of translational initiation and again via the reduced opportunity for interactions with regulatory sRNA. A rigorous assessment of these features in Mycobacerium smegmatis , however, failed to identify a consistent global trend differentiating rates of transcription and translation by leadered or leaderless transcription ( Nguyen et al, 2020 ). The observed preference for leadered transcripts in the induced response to glycopeptide antibiotic stress, and the enrichment of leaderless transcripts in those remaining unchanged following antibiotic challenge ( Table 1 ), suggests that in S. coelicolor the presence of a 5′-UTR may give more reliable dynamic control over gene product formation.…”