Project description:The vast majority of bacterial open reading frames (ORFs) are identified by automated prediction algorithms. However, these algorithms fail to identify ORFs that deviate from the canonical features of ORFs such as a length of >50 codons, and the presence of an upstream Shine-Dalgarno sequence. Here, we use ribosome profiling approaches to experimentally identify actively translated ORFs in Mycobacterium tuberculosis. Most of the ORFs we identify have not been previously described, indicating that the M. tuberculosis transcriptome is pervasively translated. Moreover, the newly described ORFs are predominantly short, with many encoding proteins of ≤50 amino acids. The codon usage of the newly discovered ORFs suggests that most are not undergoing purifying selection, and hence are unlikely to contribute to cell fitness. Nevertheless, we identify ~90 new ORFs, with a median length of 52 codons, that bear the hallmarks of purifying selection. Thus, our data suggest that pervasive translation of short ORFs serves as a rich source for the evolution of new functional proteins
Project description:Open reading frame (ORF) boundaries in bacterial genomes have largely been drawn by gene prediction algorithms. However, these algorithms often fail to predict ORFs with non-canonical features, including those that are short, overlapping, or lack 5’ UTRs. Recent developments in genome-scale mapping of translation have facilitated the empirical identification of open reading frames (ORFs). Here, we use ribosome profiling approaches to map initiating and elongating ribosomes in Mycobacterium tuberculosis. Thus, we identify over 1,000 novel ORFs, revealing that much of the M. tuberculosis genome encodes proteins in overlapping reading frames, and/or on both strands. Most of the novel ORFs are short (sORFs), impeding their identification by traditional methods. The strong codon bias that characterizes annotated mycobacterial ORFs is not evident in the aggregate novel sORFs, and hence most are unlikely to encode functional proteins. Thus, our data suggest that bacterial transcriptomes are subject to pervasive translation that occurs as a result of the relatively low specificity requirements of initiating ribosomes. We speculate that the inefficiency of expressing spurious sORFs may be offset by positive contributions to M. tuberculosis biology through cis and trans regulatory activities of a small subset.
Project description:We present the data obtained from high-resolution ribosome profiling analysis of Mycobacterium tuberculosis grown under standard conditions (log phase) and cells subjected to oxidative stress (50 µM cumene hydroperoxide) and pH stress (pH 4.5). Our data shows pervasive ribosome pausing in the M. tb translatome. A large number of genes show very high pause immediately downstream to the translation start site. Moreover, serines and alanines in the E site of the ribosome exhibit highest pause scores.
Project description:The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin-Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in Enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify cleavage targets for 12 VapCs from M. tuberculosis by applying UV-crosslinking and deep sequencing. Remarkably, these VapCs are all endoribo-nucleases that cleave RNA targets that are essential for decoding at the ribosomal A-site. Eleven VapCs cleave specific tRNAs while one exhibits SRL cleavage activity. These findings suggest that multiple vapBC modules contribute to the survival of M. tuberculosis in its human host by reducing the level of translation.
Project description:This SuperSeries is composed of the following subset Series: GSE36341: mRNA degradation in Mycobacterium tuberculosis under aerobic conditions GSE36342: mRNA degradation in Mycobacterium smegmatis under aerobic conditions GSE36343: mRNA degradation in Mycobacterium tuberculosis during cold and hypoxic stress GSE36344: mRNA degradation in Mycobacterium tuberculosis with DosR ectopically induced Refer to individual Series
Project description:Comparison of gene expression profile of the whiB4 mutant strain of Mycobacterium tuberculosis with the wild type Mycobacterium tuberculosis H37RV Mtb WhiB4 mutant mRNA was compared with the mRNA of wtMtb H37RV under aerobic conditons
Project description:To help elucidate the effect of MazF-mt9 (Rv2063A) toxin expression on translation, we generated proteomic profiles of Mycobacterium tuberculosis H37Rv cells ectopically expressing MazF-mt9
Project description:Transcriptional profiling of Mycobacterium tuberculosis H37Rv strains comparing control DMSO treated strains with Lupulone treated strains. Goal was to determine the effects of Lupulone against Mycobacterium tuberculosis H37Rv strains.