Global Regulation by CsrA and Its RNA Antagonists

Romeo, Tony; Babitzke, Paul

doi:10.1128/9781683670247.ch19

Cited by 19 publications

(37 citation statements)

References 114 publications

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“…Such obstacles include the 30S ribosomal subunit bound to an SD-like site far upstream of the translation start site in one case (39). UTRs can also contain binding sites for the global regulator CsrA, which can both promote and prevent mRNA decay in E. coli (40).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…With regard to post-transcriptional regulation, mutants with slightly changed CsrA binding efficiency (CsrA1, CsrB) also showed no significant population noise differences compared to the 'wild-type reporter' S strain. However, mutants with strongly altered CsrA binding (CsrA2, ΔLexA/CsrA2) and specifically mutations that increase CsrA abundance in the cell by changing the number of binding partners for CsrA [47] (CsrBC) showed a strong reduction in population noise in CFP, indicating the importance of CsrA availability for cel population noise. With regard to the CsrA2 mutant, a further factor might play an important role: The CsrA binding site lies within a stem loop.…”

Section: Discussionmentioning

confidence: 99%

“…Here, different possible mechanisms can come into play. CsrA itself was shown to be a global negative regulator of transcription [57], that can cause transcription attenuation by mediating Rho-dependent termination [47] and furthermore regulates mRNA stability [47,57]. In addition, CsrA could act indirectly, by affecting the abundance of other global proteins.…”

Section: Discussionmentioning

confidence: 99%

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Gene expression noise in a complex artificial toxin expression system

et al. 2020

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Gene expression is an intrinsically stochastic process. Fluctuations in transcription and translation lead to cell-to-cell variations in mRNA and protein levels affecting cellular function and cell fate. Here, using fluorescence time-lapse microscopy, we quantify noise dynamics in an artificial operon in Escherichia coli, which is based on the native operon of ColicinE2, a toxin. In the natural system, toxin expression is controlled by a complex regulatory network; upon induction of the bacterial SOS response, ColicinE2 is produced (cea gene) and released (cel gene) by cell lysis. Using this ColicinE2-based operon, we demonstrate that upon induction of the SOS response noise of cells expressing the operon is significantly lower for the (mainly) transcriptionally regulated gene cea compared to the additionally post-transcriptionally regulated gene cel. Likewise, we find that mutations affecting the transcriptional regulation by the repressor LexA do not significantly alter the population noise, whereas specific mutations to post-transcriptionally regulating units, strongly influence noise levels of both genes. Furthermore, our data indicate that global factors, such as the plasmid copy number of the operon encoding plasmid, affect gene expression noise of the entire operon. Taken together, our results provide insights on how noise in a native toxin-producing operon is controlled and underline the importance of post-transcriptional regulation for noise control in this system. OPEN ACCESSCitation: Goetz A, Mader A, von Bronk B, Weiss AS, Opitz M (2020) Gene expression noise in a complex artificial toxin expression system. PLoS ONE 15(1): e0227249. https://doi.org/10.

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“…One of these networks is the carbon storage regulatory (Csr) system in Escherichia coli . Conserved across many bacterial species, this network regulates expression of genes involved in carbon metabolism, iron homeostasis, motility, biofilm formation, the stringent response, cyclic-di-GMP synthesis, quorum sensing, and many other processes for survival and virulence ( 1 – 3 ). Recent integrated transcriptomic studies demonstrated that the Csr system controls expression of hundreds of genes in E. coli including numerous regulatory and stress response pathways ( 4 – 6 ).…”

Section: Introductionmentioning

confidence: 99%

CsrA-Mediated Translational Activation of ymdA Expression in Escherichia coli

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Poly

Lai

et al. 2020

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The sequence-specific RNA-binding protein CsrA is the central component of the conserved global regulatory Csr system. In Escherichia coli, CsrA regulates many cellular processes, including biofilm formation, motility, carbon metabolism, iron homeostasis, and stress responses. Such regulation often involves translational repression by CsrA binding to an mRNA target, thereby inhibiting ribosome binding. While CsrA also extensively activates gene expression, no detailed mechanism for CsrA-mediated translational activation has been demonstrated. An integrated transcriptomic study identified ymdA as having the strongest CsrA-mediated activation across the E. coli transcriptome. Here, we determined that CsrA activates ymdA expression posttranscriptionally. Gel mobility shift, footprint, toeprint, and in vitro coupled transcription-translation assays identified two CsrA binding sites in the leader region of the ymdA transcript that are critical for translational activation. Reporter fusion assays confirmed that CsrA activates ymdA expression at the posttranscriptional level in vivo. Furthermore, loss of binding at either of the two CsrA binding sites abolished CsrA-dependent activation. mRNA half-life studies revealed that CsrA also contributes to stabilization of ymdA mRNA. RNA structure prediction revealed an RNA hairpin upstream of the ymdA start codon that sequesters the Shine-Dalgarno (SD) sequence, which would inhibit ribosome binding. This hairpin also contains one of the two critical CsrA binding sites, with the other site located just upstream. Our results demonstrate that bound CsrA destabilizes the SD-sequestering hairpin such that the ribosome can bind and initiate translation. Since YmdA represses biofilm formation, CsrA-mediated activation of ymdA expression may repress biofilm formation under certain conditions. IMPORTANCE The Csr system of E. coli controls gene expression and physiology on a global scale. CsrA protein, the central component of this system, represses translation initiation of numerous genes by binding to target transcripts, thereby competing with ribosome binding. Variations of this mechanism are so common that CsrA is sometimes called a translational repressor. Although CsrA-mediated activation mechanisms have been elucidated in which bound CsrA inhibits RNA degradation, no translation activation mechanism has been defined. Here, we demonstrate that CsrA binding to two sites in the 5′ untranslated leader of ymdA mRNA activates translation by destabilizing a structure that otherwise prevents ribosome binding. The extensive role of CsrA in activating gene expression suggests the common occurrence of similar activation mechanisms.

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Global Regulation by CsrA and Its RNA Antagonists

Cited by 19 publications

References 114 publications

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

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

Gene expression noise in a complex artificial toxin expression system

CsrA-Mediated Translational Activation of ymdA Expression in Escherichia coli

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