Project description:Analysis of the contribution of the post-transcriptional regulator CsrA to translation during exponential growth in Escherichia coli Overall design: 5 biological replicates of ribosome protected fragments and paired RNA samples from wild type and csrA::kan mutant strains were analyzed
Project description:Analysis of the contribution of the post-transcriptional regulator CsrA to translation during exponential growth in Escherichia coli Overall design: 4 biological replicates of ribosome protected fragments and paired RNA samples from wild type and csrA:: mutant strains in mLPM and LB were analyzed
Project description:Bacterial global post-transcriptional regulators execute hundreds of interactions with targets that display varying molecular features while retaining specificity. Herein, we develop, validate, and apply a biophysical, statistical thermodynamic model of canonical target mRNA interactions with the CsrA global post-transcriptional regulator to understand the molecular features that contribute to target regulation. Altogether, we model interactions of CsrA with a pool of 236 mRNA: 107 are experimentally regulated by CsrA and 129 are suspected interaction partners. Guided by current understanding of CsrA-mRNA interactions, we incorporate (i) mRNA nucleotide sequence, (ii) cooperativity of CsrA-mRNA binding, and (iii) minimization of mRNA structural changes to identify an ensemble of likely binding sites and their free energies. The regulatory impact of bound CsrA on mRNA translation is determined with the RBS calculator. Predicted regulation of 66 experimentally regulated mRNAs adheres to the principles of canonical CsrA-mRNA interactions; the remainder implies that other, diverse mechanisms may underlie CsrA-mRNA interaction and regulation. Importantly, results suggest that this global regulator may bind targets in multiple conformations, via flexible stretches of overlapping predicted binding sites. This novel observation expands the notion that CsrA always binds to its targets at specific consensus sequences.
Project description:CsrA is a post-transcriptional regulatory protein that is widely distributed among bacteria. This protein influences bacterial lifestyle decisions by binding to the 5' untranslated and/or early coding regions of mRNA targets, causing changes in translation initiation, RNA stability, and/or transcription elongation. Here, we assess the contribution of CsrA to gene expression in Escherichia coli on a global scale. UV crosslinking immunoprecipitation and sequencing (CLIP-seq) identify RNAs that interact directly with CsrA in vivo, while ribosome profiling and RNA-seq uncover the impact of CsrA on translation, RNA abundance, and RNA stability. This combination of approaches reveals unprecedented detail about the regulatory role of CsrA, including novel binding targets and physiological roles, such as in envelope function and iron homeostasis. Our findings highlight the integration of CsrA throughout the E. coli regulatory network, where it orchestrates vast effects on gene expression.
Project description:The Lyme disease spirochete Borrelia burgdorferi lacks the transcriptional cascade control of flagellar protein synthesis common to other bacteria. Instead, it relies on a post-transcriptional mechanism to control its flagellar synthesis. The underlying mechanism of this control remains elusive. A recent study reported that the increased level of BB0184 (CsrA(Bb); a homologue of carbon storage regulator A) substantially inhibited the accumulation of FlaB, the major flagellin protein of B. burgdorferi. In this report, we deciphered the regulatory role of CsrA(Bb) on FlaB synthesis and the mechanism involved by analysing two mutants, csrA(Bb)(-) (a deletion mutant of csrA(Bb)) and csrA(Bb)(+) (a mutant conditionally overexpressing csrA(Bb)). We found that FlaB accumulation was significantly inhibited in csrA(Bb)(+) but was substantially increased in csrA(Bb)(-) . In contrast, the levels of other flagellar proteins remained unchanged. Cryo-electron tomography and immuno-fluorescence microscopic analyses revealed that the altered synthesis of CsrA(Bb) in these two mutants specifically affected flagellar filament length. The leader sequence of flaB transcript contains two conserved CsrA-binding sites, with one of these sites overlapping the Shine-Dalgarno sequence. We found that CsrA(Bb) bound to the flaB transcripts via these two binding sites, and this binding inhibited the synthesis of FlaB at the translational level. Taken together, our results indicate that CsrA(Bb) specifically regulates the periplasmic flagellar synthesis by inhibiting translation initiation of the flaB transcript.
Project description:Expression profiles of wild-type and SgrR mutant E. coli strains under aMG and 2-DG-induced stress. Expression profiles of E. coli overexpressing SgrS sRNA. Illumina RNA-Seq of total RNA extracted from wild-type, SgrR/SgrS mutant and SgrS overexpressing E. coli strains grown in different conditions.
Project description:The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.
Project description:Analysis of the contribution of the post-transcriptional regulator CsrA to RNA stability during exponential growth in Escherichia coli Overall design: 2 replicate time courses with 5 time points from wild type and csrA::kan mutant strains were analyzed