Transcription initiation in mycobacteria: a biophysical perspective.
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ABSTRACT:
SUBMITTER: Boyaci H
PROVIDER: S-EPMC7549741 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Each genome encodes some codons more frequently than their synonyms (codon usage bias), but codons are also arranged more frequently into specific pairs (codon pair bias). Recoding viral genomes and yeast or bacterial genes with non-optimal codon pairs has been shown to decrease gene expression. Gene expression is thus importantly regulated not only by the use of particular codons but also by their proper juxtaposition. We therefore hypothesized that non-optimal codon pairing could likewise attenuate Mycobacterium tuberculosis (Mtb) genes. We explored the role of codon pair bias by recoding Mtb genes (rpoB, mmpL3, and ndh) and assessing their expression in the closely related and tractable model organism M. smegmatis. To our surprise, recoding caused the expression of multiple smaller protein isoforms from all three genes. We confirmed that these smaller proteins were not due to protein degradation but instead issued from new transcription initiation sites positioned within the open reading frame. New transcripts gave rise to intragenic translation initiation sites, which, in turn, led to the expression of smaller proteins. We next identified the nucleotide changes associated with these new sites of transcription and translation. Our results demonstrated that apparently benign, synonymous changes can drastically alter gene expression in mycobacteria. More generally, our work expands our understanding of the codon-level parameters that control translation and transcription initiation. IMPORTANCE Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb. We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is one of the first reports that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.
| S-EPMC10653884 | biostudies-literature
Project description:The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2?Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76?Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP ?-subunit C-terminal domain (?CTD) with DNA, and we provide evidence that the ?CTD may play a role in Mtb transcription regulation. Our results reveal the structure of an Actinobacteria-unique insert of the RNAP ?' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm ?A, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.
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Project description:BackgroundMycobacteria produce two unique families of cytoplasmic polymethylated polysaccharides -- the methylglucose lipopolysaccharides (MGLPs) and the methylmannose polysaccharides (MMPs) -- the physiological functions of which are still poorly defined. Towards defining the roles of these polysaccharides in mycobacterial physiology, we generated knock-out mutations of genes in their putative biosynthetic pathways.Methodology/principal findingsWe report here on the characterization of the Rv1208 protein of Mycobacterium tuberculosis and its ortholog in Mycobacterium smegmatis (MSMEG_5084) as the enzymes responsible for the transfer of the first glucose residue of MGLPs. Disruption of MSMEG_5084 in M. smegmatis resulted in a dramatic decrease in MGLP synthesis directly attributable to the almost complete abolition of glucosyl-3-phosphoglycerate synthase activity in this strain. Synthesis of MGLPs in the mutant was restored upon complementation with wild-type copies of the Rv1208 gene from M. tuberculosis or MSMEG_5084 from M. smegmatis.Conclusions/significanceThis is the first evidence linking Rv1208 to MGLP biosynthesis. Thus, the first step in the initiation of MGLP biosynthesis in mycobacteria has been defined, and subsequent steps can be inferred.
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Project description:Unexpected experimental outcomes led us to hypothesize that synonymously recoding segments of the M. tuberculosis (Mtb) rpoB gene (rv0667) caused the translation of many smaller protein isoforms from translation initiations sites within the recoded open reading frame (ORF). To test this hypothesis, we labeled the N-termini of Mtb rpoB expressed heterologously in the closely related model system M. smegmatis and identified peptides of rpoB carrying the N-terminal label by mass spectrometry. We found an increase in the detection of N-terminally labeled peptides that mapped to recoded regions of the rpoB gene variants, and thus inferred that recoding was linked to the generation of translation initiation sites within the ORF. We also observed a smaller number of N-terminally labeled peptides mapping to the middle of the wild-type ORF, which we interpreted to indicate that some intragenic translation initiation could possibly occur in the native gene or, more likely, arise from heterologous gene expression.
2022-05-27 | MSV000089557 | MassIVE
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Project description:SummaryTranscription factories are nuclear domains where gene transcription takes place although the molecular basis for their formation and maintenance are unknown. In this study, we explored how the properties of chromatin as a polymer may contribute to the structure of transcription factories. We found that transcriptional active chromatin contains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Single fibre analysis showed that this modification spans the entire body of the gene. Furthermore, H4K16ac genes cluster in regions up to 500 Kb alternating active and inactive chromatin. The introduction of H4K16ac in chromatin induces stiffness in the chromatin fibre. The result of this change in flexibility is that chromatin could behave like a multi-block copolymer with repetitions of stiff-flexible (active-inactive chromatin) components. Copolymers with such structure self-organize through spontaneous phase separation into microdomains. Consistent with such model H4K16ac chromatin form foci that associates with nascent transcripts. We propose that transcription factories are the result of the spontaneous concentration of H4K16ac chromatin that are in proximity, mainly in cis.
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