Helen Yakhnin

CsrA activates flhDC expression by protecting flhDC mRNA from RNase E‐mediated cleavage

Csr is a conserved global regulatory system that controls expression of several hundred Escherichia coli genes. CsrA protein represses translation of numerous genes by binding to mRNA and inhibiting ribosome access. CsrA also activates gene expression, although an activation mechanism has not been reported. CsrA activates flhDC expression, encoding the master regulator of flagellum biosynthesis and chemotaxis, by stabilizing the mRNA. Computer modelling, gel mobility shift and footprint analyses identified two CsrA binding sites extending from positions 1–12 (BS1) and 44–55 (BS2) of the 198 nt flhDC leader transcript. flhD′–′lacZ expression was reduced by mutations in csrA and/or the CsrA binding sites. The position of BS1 suggested that bound CsrA might inhibit 5′ end‐dependent RNase E cleavage of flhDC mRNA. Consistent with this hypothesis, CsrA protected flhDC leader RNA from RNase E cleavage in vitro and protection depended on BS1 and BS2. Primer extension studies identified flhDC decay intermediates in vivo that correspond to in vitro RNase E cleavage sites. Deletion of these RNase E cleavage sites resulted in increased flhD′–′lacZ expression. Data from mRNA decay studies and quantitative primer extension assays support a model in which bound CsrA activates flhDC expression by inhibiting the 5′ end‐dependent RNase E cleavage pathway.

Integration of a complex regulatory cascade involving the SirA/BarA and Csr global regulatory systems that controls expression of the Salmonella SPI‐1 and SPI‐2 virulence regulons through HilD

Salmonella pathogenicity islands 1 and 2 (SPI‐1 and SPI‐2) play key roles in the pathogenesis of Salmonella enterica. Previously, we showed that when Salmonella grows in Luria–Bertani medium, HilD, encoded in SPI‐1, first induces the expression of hilA, located in SPI‐1, and subsequently of the ssrAB operon, located in SPI‐2. These genes code for HilA and the SsrA/B two‐component system, the positive regulators of the SPI‐1 and SPI‐2 regulons respectively. In this study, we demonstrate that CsrA, a global regulatory RNA binding protein, post‐transcriptionally regulates hilD expression by directly binding near the Shine–Dalgarno and translation initiation codon sequences of the hilD mRNA, preventing its translation and leading to its accelerated turnover. Negative regulation is counteracted by the global SirA/BarA two‐component system, which directly activates the expression of CsrB and CsrC, two non‐coding regulatory RNAs that sequester CsrA, thereby preventing it from binding to its target mRNAs. Our results illustrate the integration of global and specific regulators into a multifactorial regulatory cascade controlling the expression of virulence genes acquired by horizontal transfer events.

CsrA of Bacillus subtilis regulates translation initiation of the gene encoding the flagellin protein (hag) by blocking ribosome binding

The global regulatory Csr (carbon storage regulator) and the homologous Rsm (repressor of secondary metabolites) systems of Gram‐negative bacteria typically consist of an RNA‐binding protein (CsrA/RsmA) and at least one sRNA that functions as a CsrA antagonist. CsrA modulates gene expression post‐transcriptionally by regulating translation initiation and/or mRNA stability of target transcripts. While Csr has been extensively studied in Gram‐negative bacteria, until now Csr has not been characterized in any Gram‐positive organism. csrA of Bacillus subtilis is the last gene of a flagellum biosynthetic operon. In addition to the previously identified σD‐dependent promoter that controls expression of the entire operon, a σA‐dependent promoter was identified that temporally controls expression of the last two genes of the operon (fliW‐csrA); expression peaks 1 h after cell growth deviates from exponential phase. hag, the gene encoding flagellin, was identified as a CsrA‐regulated gene. CsrA was found to repress hag′–′lacZ expression, while overexpression of csrA reduces cell motility. In vitro binding studies identified two CsrA binding sites in the hag leader transcript, one of which overlaps the hag Shine–Dalgarno sequence. Toeprint and cell‐free translation studies demonstrate that bound CsrA prevents ribosome binding to the hag transcript, thereby inhibiting translation initiation and Hag synthesis.

CsrA–FliW interaction governs flagellin homeostasis and a checkpoint on flagellar morphogenesis in Bacillus subtilis

CsrA is a widely distributed RNA binding protein that regulates translation initiation and/or mRNA stability of target transcripts. CsrA activity is antagonized by sRNA(s) containing multiple CsrA binding sites in several Gram‐negative bacterial species. Here we discover FliW, the first protein antagonist of CsrA activity that constitutes a partner switching mechanism to control flagellin synthesis in the Gram‐positive organism Bacillus subtilis. Following the flagellar assembly checkpoint of hook completion, secretion of flagellin (Hag) releases FliW protein from a FliW‐Hag complex. FliW then binds to CsrA and relieves CsrA‐mediated translational repression of hag for flagellin synthesis concurrent with filament assembly. Thus, flagellin homeostatically restricts its own translation. Homeostatic autoregulation may be a general mechanism to precisely control structural subunits required at specific times and in finite amounts such as those involved in the assembly of flagella, type III secretion machines and pili. Finally, phylogenetic analysis suggests that CsrA, a highly pleiotropic virulence regulator in many bacterial pathogens, had an ancestral role in flagellar assembly and evolved to co‐regulate various cellular processes with motility.

The trp RNA-Binding Attenuation Protein of Bacillus subtilis Regulates Translation of the Tryptophan Transport Gene trpP (yhaG) by Blocking Ribosome Binding

Expression of the Bacillus subtilis tryptophan biosynthetic genes (trpEDCFBA and pabA [trpG]) is regulated in response to tryptophan by TRAP, the trp RNA-binding attenuation protein. TRAP-mediated regulation of the tryptophan biosynthetic genes includes a transcription attenuation and two distinct translation control mechanisms. TRAP also regulates translation of trpP (yhaG), a single-gene operon that encodes a putative tryptophan transporter. Its translation initiation region contains triplet repeats typical of TRAP-regulated mRNAs. We found that regulation of trpP and pabA is unaltered in a rho mutant strain. Results from filter binding and gel mobility shift assays demonstrated that TRAP binds specifically to a segment of the trpP transcript that includes the untranslated leader and translation initiation region. While the affinities of TRAP for the trpP and pabA transcripts are similar, TRAP-mediated translation control of trpP is much more extensive than for pabA. RNA footprinting revealed that the trpP TRAP binding site consists of nine triplet repeats (five GAG, three UAG, and one AAG) that surround and overlap the trpP Shine-Dalgarno (S-D) sequence and translation start codon. Results from toeprint and RNA-directed cell-free translation experiments indicated that tryptophan-activated TRAP inhibits TrpP synthesis by preventing binding of a 30S ribosomal subunit. Taken together, our results establish that TRAP regulates translation of trpP by blocking ribosome binding. Thus, TRAP coordinately regulates tryptophan synthesis and transport by three distinct mechanisms: attenuation transcription of the trpEDCFBA operon, promoting formation of the trpE S-D blocking hairpin, and blocking ribosome binding to the pabA and trpP transcripts.

Complex regulation of the global regulatory gene csrA: CsrA-mediated translational repression, transcription from five promoters by Eσ70 and EσS, and indirect transcriptional activation by CsrA

CsrA of Escherichia coli is an RNA‐binding protein that globally regulates gene expression by repressing translation and/or altering the stability of target transcripts. Here we explored mechanisms that control csrA expression. Four CsrA binding sites were predicted upstream of the csrA initiation codon, one of which overlapped its Shine–Dalgarno sequence. Results from gel shift, footprint, toeprint and in vitro translation experiments indicate that CsrA binds to these four sites and represses its own translation by directly competing with 30S ribosomal subunit binding. Experiments were also performed to examine transcription of csrA. Primer extension, in vitro transcription and in vivo expression studies identified two σ70‐dependent (P2 and P5) and two σS‐dependent (P1 and P3) promoters that drive transcription of csrA. Additional primer extension studies identified a fifth csrA promoter (P4). Transcription from P3, which is indirectly activated by CsrA, is primarily responsible for increased csrA expression as cells transition from exponential to stationary‐phase growth. Taken together, our results indicate that regulation of csrA expression occurs by a variety of mechanisms, including transcription from multiple promoters by two sigma factors, indirect activation of its own transcription, as well as direct repression of its own translation.

Function of the Bacillus subtilis transcription elongation factor NusG in hairpin-dependent RNA polymerase pausing in the trp leader

NusA and NusG are transcription elongation factors that bind to RNA polymerase (RNAP) after σ subunit release. Escherichia coli NusA (NusAEc) stimulates intrinsic termination and RNAPEc pausing, whereas NusGEc promotes Rho-dependent termination and pause escape. Both Nus factors also participate in the formation of RNAPEc antitermination complexes. We showed that Bacillus subtilis NusA (NusABs) stimulates intrinsic termination and RNAPBs pausing at U107 and U144 in the trpEDCFBA operon leader. Pausing at U107 and U144 participates in the transcription attenuation and translational control mechanisms, respectively, presumably by providing additional time for trp RNA-binding attenuation protein (TRAP) to bind to the nascent trp leader transcript. Here, we show that NusGBs causes modest pause stimulation at U107 and dramatic pause stimulation at U144. NusABs and NusGBs act synergistically to increase the U107 and U144 pause half-lives. NusGBs-stimulated pausing at U144 requires RNAPBs, whereas NusABs stimulates pausing of RNAPBs and RNAPEc at the U144 and E. coli his pause sites. Although NusGEc does not stimulate pausing at U144, it competes with NusGBs-stimulated pausing, suggesting that both proteins bind to the same surface of RNAPBs. Inactivation of nusG results in the loss of RNAP pausing at U144 in vivo and elevated trp operon expression, whereas plasmid-encoded NusG complements the mutant defects. Overexpression of nusG reduces trp operon expression to a larger extent than overexpression of nusA.

The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis regulates translation initiation of ycbK, a gene encoding a putative efflux protein, by blocking ribosome binding

Expression of the Bacillus subtilis tryptophan biosynthetic genes trpEDCFBA and trpG, as well as a putative tryptophan transport gene (trpP), are regulated in response to tryptophan by the trp RNA‐binding attenuation protein (TRAP). TRAP regulates expression of these genes by transcription attenuation and translation control mechanisms. Here we show that TRAP also regulates translation of ycbK, a gene that encodes a protein with similarities to known efflux proteins. As a likely TRAP‐binding site consisting of 11 NAG repeats overlaps the ycbK translation initiation region, experiments were carried out to determine whether TRAP regulates translation of ycbK. TRAP was observed to regulate expression of a ycbK′–′lacZ translational fusion 20‐fold in response to tryptophan. Binding studies indicated that TRAP binds to the ycbK transcript with high affinity and specificity. Footprint studies revealed that the central seven triplet repeats were protected by bound TRAP, while toeprint results suggest that nine triplet repeats contribute to TRAP binding. Additional toeprint and in vitro translation analyses demonstrated that bound TRAP regulates YcbK synthesis by blocking ribosome binding. We also identified two dipeptide coding minigenes between the Shine‐Dalgarno sequence and start codon of ycbK. Expression of one of the minigenes modestly interfered with translation of ycbK.

Gel Mobility Shift Assays to Detect Protein–RNA Interactions

The gel mobility shift assay is a powerful technique for detecting and quantifying protein-RNA interactions. While other techniques such as filter binding and isothermal titration calorimetry (ITC) are available for quantifying protein-RNA interactions, gel shift analysis provides the added advantage that you can visualize the protein-RNA complexes. In the gel shift assay, protein-RNA complexes are typically separated from the unbound RNA using native polyacrylamide gels in Tris/borate/EDTA buffer, although an alternative Tris-glycine buffering system is superior in many situations. Here, we describe both gel shift methods, along with strategies to improve separation of protein-RNA complexes from free RNA, which can be a particular challenge for small RNA binding proteins.

Global role of the bacterial post-transcriptional regulator CsrA revealed by integrated transcriptomics

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.The RNA-binding protein CsrA regulates the expression of hundreds of bacterial genes. Here, Potts et al. use several approaches to assess the contribution of CsrA to global gene expression in E. coli, revealing new binding targets and physiological roles such as in envelope function and iron homeostasis.