Alexander V. 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.

NusA-stimulated RNA polymerase pausing and termination participates in the Bacillus subtilis trp operon attenuation mechanism in vitro

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translation control mechanisms. Both mechanisms require the binding of tryptophan-activated TRAP to the 11 (G/U)AG-repeat segment in the trp leader transcript. To promote termination, TRAP must bind to the nascent RNA before the antiterminator structure forms. Because only 20 nucleotides separate the TRAP-binding site from the 3′ end of the antiterminator, TRAP has a short time frame to control this regulatory decision. Synchronization of factor binding and/or RNA folding with the RNA polymerase position is a major challenge in all attenuation mechanisms. Because RNA polymerase pausing allows this synchronization in many attenuation mechanisms, we performed experiments in vitro to determine whether pausing participates in the B. subtilis trp attenuation mechanism. We identified two NusA-stimulated pause sites in the trp leader region. Formation of pause hairpins participates in pausing at both positions. The first pause occurred at the nucleotide just preceding the critical overlap between the alternative antiterminator and terminator structures. TRAP binding to transcripts containing preexisting pause complexes releases RNA polymerase, suggesting that pausing provides additional time for TRAP to bind and promote termination. The second pause is downstream from the trp leader termination point, raising the possibility that this pause event participates in the trpE translation control mechanism. NusA also increases the efficiency of termination in the trp leader region and shifts termination one nucleotide upstream. Finally, NusA-stimulated termination is cooperative, suggesting that binding of multiple NusA molecules influences termination.

Effects of Mutations in the l-Tryptophan Binding Pocket of the trp RNA-binding Attenuation Protein ofBacillus subtilis

The Bacillus subtilis tryptophan biosynthetic genes are regulated by the trp RNA-binding attenuation protein (TRAP). Cooperative binding ofl-tryptophan activates TRAP so that it can bind to RNA. The crystal structure revealed that l-tryptophan forms nine hydrogen bonds with various amino acid residues of TRAP. We performed site-directed mutagenesis to determine the importance of several of these hydrogen bonds in TRAP activation. We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions. Tryptophan binding mutations were identified in vivo having unchanged, reduced, or completely eliminated repression activity. Several of thein vivo defective TRAP mutants exhibited reduced affinity for tryptophan in vitro but did not interfere with RNA binding at saturating tryptophan concentrations. However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulation, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP. One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function. Another mutant protein exhibited tryptophan-independent RNA binding activity. We also found that trp leader RNA increases the affinity of TRAP for tryptophan.

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.

Mechanism of NusG-stimulated pausing, hairpin-dependent pause site selection and intrinsic termination at overlapping pause and termination sites in the Bacillus subtilis trp leader

The Bacillus subtilis trpEDCFBA operon is regulated by TRAP‐dependent transcription attenuation and translation repression mechanisms. Previous results showed that NusA and NusG cooperatively stimulate RNA polymerase pausing at U107 and U144 in the trp leader, and that NusG is required for pausing at U144 in vivo. Pausing at U107 and U144 participate in the attenuation and translation repression mechanisms, respectively, by providing additional time for TRAP binding. The intrinsic trp leader terminator overlaps the hairpin‐dependent U144 pause site. Here, we conducted a systematic mutational analysis of the terminator/pause region. Deletion of the hairpin reduced pausing but did not affect pause site selection. Thus, hairpin‐stimulated pausing is a more appropriate term than hairpin‐dependent pausing for this pause site. In contrast, minor changes to the hairpin abolished termination. Sequences in the U‐rich/T‐rich tract following the hairpin affected termination and pausing differentially. The distance between the hairpin and the 3′ end of the RNA dictates the position of termination, whereas the sequence downstream from the hairpin is responsible for pause site selection. NusA was found to increase both pausing and termination by reducing the rate of transcription. We also found that NusG‐stimulated pausing is sequence specific and that NusG does not affect termination.

NusG is a Sequence-specific RNA Polymerase Pause Factor that Binds to the Non-template DNA Within the Paused Transcription Bubble

NusG, referred to as Spt5 in archaeal and eukaryotic organisms, is the only transcription factor conserved in all three domains of life. This general transcription elongation factor binds to RNA polymerase (RNAP) soon after transcription initiation and dissociation of the RNA polymerase σ factor. Escherichia coli NusG increases transcription processivity by suppressing RNAP pausing, whereas Bacillus subtilis NusG dramatically stimulates pausing at two sites in the untranslated leader of the trpEDCFBA operon. These two regulatory pause sites participate in transcription attenuation and translational control mechanisms, respectively. Here we report that B. subtilis NusG makes sequence-specific contacts with a T-rich sequence in the non-template DNA (ntDNA) strand within the paused transcription bubble. NusG protects T residues of the recognition sequence from permanganate oxidation, and these T residues increase the affinity of NusG to the elongation complex. Binding of NusG to RNAP does not require interaction with RNA. These results indicate that bound NusG prevents forward movement of RNA polymerase by simultaneously contacting RNAP and the ntDNA strand. Mutational studies indicate that amino acid residues of two short regions within the NusG N-terminal domain are primarily responsible for recognition of the trp operon pause signals. Structural modeling indicates that these two regions are adjacent to each another in the protein. We propose that recognition of specific sequences in the ntDNA and stimulation of RNAP pausing is a conserved function of NusG-like transcription factors.

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.

NusA-dependent transcription termination prevents misregulation of global gene expression

Intrinsic transcription terminators consist of an RNA hairpin followed by a U-rich tract, and these signals can trigger termination without the involvement of additional factors. Although NusA is known to stimulate intrinsic termination in vitro, the in vivo targets and global impact of NusA are not known because it is essential for viability. Using genome-wide 3′ end-mapping on an engineered Bacillus subtilis NusA depletion strain, we show that weak suboptimal terminators are the principle NusA substrates. Moreover, a subclass of weak non-canonical terminators was identified that completely depend on NusA for effective termination. NusA-dependent terminators tend to have weak hairpins and/or distal U-tract interruptions, supporting a model in which NusA is directly involved in the termination mechanism. Depletion of NusA altered global gene expression directly and indirectly via readthrough of suboptimal terminators. Readthrough of NusA-dependent terminators caused misregulation of genes involved in essential cellular functions, especially DNA replication and metabolism. We further show that nusA is autoregulated by a transcription attenuation mechanism that does not rely on antiterminator structures. Instead, NusA-stimulated termination in its 5′ UTR dictates the extent of transcription into the operon, thereby ensuring tight control of cellular NusA levels.