Premature termination of transcription is shaped by Rho and translated uORFS in Mycobacterium tuberculosis

Little is known about the decisions behind transcription elongation versus termination in the human pathogen Mycobacterium tuberculosis (M.TB). By applying Term-seq to M.TB we found that the majority of transcription termination is premature and associated with translated regions, i.e., within previously annotated or newly identified open reading frames. Computational predictions and Term-seq analysis, upon depletion of termination factor Rho, suggests that Rho-dependent transcription termination dominates all transcription termination sites (TTS), including those associated with regulatory 5′ leaders. Moreover, our results suggest that tightly coupled translation, in the form of overlapping stop and start codons, may suppress Rho-dependent termination. This study provides detailed insights into novel M.TB cis-regulatory elements, where Rho-dependent, conditional termination of transcription and translational coupling together play major roles in gene expression control. Our findings contribute to a deeper understanding of the fundamental regulatory mechanisms that enable M.TB adaptation to the host environment offering novel potential points of intervention

Genome-wide mapping of transcriptional start sites defines an extensive leaderless transcriptome in Mycobacterium tuberculosis.

Deciphering physiological changes that mediate transition of Mycobacterium tuberculosis between replicating and nonreplicating states is essential to understanding how the pathogen can persist in an individual host for decades. We have combined RNA sequencing (RNA-seq) of 5' triphosphate-enriched libraries with regular RNA-seq to characterize the architecture and expression of M. tuberculosis promoters. We identified over 4,000 transcriptional start sites (TSSs). Strikingly, for 26% of the genes with a primary TSS, the site of transcriptional initiation overlapped with the annotated start codon, generating leaderless transcripts lacking a 5' UTR and, hence, the Shine-Dalgarno sequence commonly used to initiate ribosomal engagement in eubacteria. Genes encoding proteins with active growth functions were markedly depleted from the leaderless transcriptome, and there was a significant increase in the overall representation of leaderless mRNAs in a starvation model of growth arrest. The high percentage of leaderless genes may have particular importance in the physiology of nonreplicating M. tuberculosis

Cell-wall synthesis and ribosome maturation are co-regulated by an RNA switch in Mycobacterium tuberculosis

The success of Mycobacterium tuberculosis relies on the ability to switch between active growth and non-replicating persistence, associated with latent TB infection. Resuscitation promoting factors (Rpfs) are essential for the transition between these states. Rpf expression is tightly regulated as these enzymes are able to degrade the cell wall, and hence potentially lethal to the bacterium itself. We have identified a regulatory element in the 5' untranslated region (UTR) of rpfB. We demonstrate that this element is a transcriptionally regulated RNA switch/riboswitch candidate, which appears to be restricted to pathogenic mycobacteria, suggesting a role in virulence. We have used translation start site mapping to re-annotate the RpfB start codon and identified and validated a ribosome binding site that is likely to be targeted by an rpfB antisense RNA. Finally, we show that rpfB is co-transcribed with ksgA and ispE downstream. ksgA encodes a universally conserved methyltransferase involved in ribosome maturation and ispE encodes an essential kinase involved in cell wall synthesis. This arrangement implies co-regulation of resuscitation, cell wall synthesis and ribosome maturation via the RNA switch

The Mycobacterium tuberculosis sRNA F6 Modifies Expression of Essential Chaperonins, GroEL2 and GroES.

Almost 140 years after the identification of Mycobacterium tuberculosis as the etiological agent of tuberculosis, important aspects of its biology remain poorly described. Little is known about the role of posttranscriptional control of gene expression and RNA biology, including the role of most of the small RNAs (sRNAs) identified to date. We have carried out a detailed investigation of the M. tuberculosis sRNA F6 and shown it to be dependent on SigF for expression and significantly induced in starvation conditions in vitro and in a mouse model of infection. Further exploration of F6 using an in vitro starvation model of infection indicates that F6 affects the expression of the essential chaperonins GroEL2 and GroES. Our results point toward a role for F6 during periods of low metabolic activity typically associated with long-term survival of M. tuberculosis in human granulomas. IMPORTANCE Control of gene expression via small regulatory RNAs (sRNAs) is poorly understood in one of the most successful pathogens, Mycobacterium tuberculosis. Here, we present an in-depth characterization of the sRNA F6, including its expression in different infection models and the differential gene expression observed upon deletion of the sRNA. Our results demonstrate that deletion of F6 leads to dysregulation of the two essential chaperonins GroEL2 and GroES and, moreover, indicate a role for F6 in the long-term survival and persistence of M. tuberculosis in the human host

The mycobacterium tuberculosis sRNA F6 modifies expression of essential chaperonins, GroEL2 and GroES

Almost 140 years after the identification of Mycobacterium tuberculosis as the etiological agent of tuberculosis, important aspects of its biology remain poorly described. Little is known about the role of posttranscriptional control of gene expression and RNA biology, including the role of most of the small RNAs (sRNAs) identified to date. We have carried out a detailed investigation of the M. tuberculosis sRNA F6 and shown it to be dependent on SigF for expression and significantly induced in starvation conditions in vitro and in a mouse model of infection. Further exploration of F6 using an in vitro starvation model of infection indicates that F6 affects the expression of the essential chaperonins GroEL2 and GroES. Our results point toward a role for F6 during periods of low metabolic activity typically associated with long-term survival of M. tuberculosis in human granulomas. IMPORTANCE Control of gene expression via small regulatory RNAs (sRNAs) is poorly understood in one of the most successful pathogens, Mycobacterium tuberculosis. Here, we present an in-depth characterization of the sRNA F6, including its expression in different infection models and the differential gene expression observed upon deletion of the sRNA. Our results demonstrate that deletion of F6 leads to dysregulation of the two essential chaperonins GroEL2 and GroES and, moreover, indicate a role for F6 in the long-term survival and persistence of M. tuberculosis in the human host

Thermodynamics of Heat Shock Response

Production of heat shock proteins are induced when a living cell is exposed to a rise in temperature. The heat shock response of protein DnaK synthesis in E.coli for temperature shifts from temperature T to T plus 7 degrees, respectively to T minus 7 degrees is measured as function of the initial temperature T. We observe a reversed heat shock at low T. The magnitude of the shock increases when one increase the distance to the temperature $T_0 \approx 23^o$, thereby mimicking the non monotous stability of proteins at low temperature. Further we found that the variation of the heat shock with T quantitatively follows the thermodynamic stability of proteins with temperature. This suggest that stability related to hot as well as cold unfolding of proteins is directly implemented in the biological control of protein folding. We demonstrate that such an implementation is possible in a minimalistic chemical network.Comment: To be published in Physical Review Letter

Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis

Sequence-Based Analysis Uncovers an Abundance of Non-Coding RNA in the Total Transcriptome of Mycobacterium tuberculosis

RNA sequencing provides a new perspective on the genome of Mycobacterium tuberculosis by revealing an extensive presence of non-coding RNA, including long 5’ and 3’ untranslated regions, antisense transcripts, and intergenic small RNA (sRNA) molecules. More than a quarter of all sequence reads mapping outside of ribosomal RNA genes represent non-coding RNA, and the density of reads mapping to intergenic regions was more than two-fold higher than that mapping to annotated coding sequences. Selected sRNAs were found at increased abundance in stationary phase cultures and accumulated to remarkably high levels in the lungs of chronically infected mice, indicating a potential contribution to pathogenesis. The ability of tubercle bacilli to adapt to changing environments within the host is critical to their ability to cause disease and to persist during drug treatment; it is likely that novel post-transcriptional regulatory networks will play an important role in these adaptive responses

Noncoding RNA in Mycobacteria.

Efforts to understand the molecular basis of mycobacterial gene regulation are dominated by a protein-centric view. However, there is a growing appreciation that noncoding RNA, i.e., RNA that is not translated, plays a role in a wide variety of molecular mechanisms. Noncoding RNA comprises rRNA, tRNA, 4.5S RNA, RnpB, and transfer-messenger RNA, as well as a vast population of regulatory RNA, often dubbed "the dark matter of gene regulation." The regulatory RNA species comprise 5' and 3' untranslated regions and a rapidly expanding category of transcripts with the ability to base-pair with mRNAs or to interact with proteins. Regulatory RNA plays a central role in the bacterium's response to changes in the environment, and in this article we review emerging information on the presence and abundance of different types of noncoding RNA in mycobacteria

Challenges in defining the functional, non-coding, expressed genome of members of the Mycobacterium tuberculosis complex

A definitive transcriptome atlas for the non-coding expressed elements of the members of the Mycobacterium tuberculosis complex (MTBC) does not exist. Incomplete lists of non-coding transcripts can be obtained for some of the reference genomes (e.g. Mycobacterium tuberculosis H37Rv) but to what extent these transcripts have homologues in closely related species or even strains is not clear. This has implications for the analysis of transcriptomic data; non-coding parts of the transcriptome are often ignored in the absence of formal, reliable annotation. Here, we review the state of our knowledge of non-coding RNAs in pathogenic mycobacteria, emphasising the disparities in the information included in commonly used databases. We then proceed to review ways of combining computational solutions for predicting the non-coding transcriptome with experiments that can help refine and confirm these predictions