The N-terminal domain of RfaH plays an active role in protein fold-switching

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a β-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a β-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.

The N-terminal domain of RfaH plays an active role in protein fold-switching

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) stalled at a DNA signal known as ops. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a β-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the ops exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a β-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, most studies employ tertiary biases towards each native state, hampering the analysis of sequence-encoded interactions on fold-switching. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and also by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.

Interplay between σ region 3.2 and secondary channel factors during promoter escape by bacterial RNA polymerase

In bacterial RNA polymerase (RNAP), conserved region 3.2 of the σ subunit was proposed to contribute to promoter escape by interacting with the 5′-end of nascent RNA, thus facilitating σ dissociation. RNAP activity during transcription initiation can also be modulated by protein factors that bind within the secondary channel and reach the enzyme active site. To monitor the kinetics of promoter escape in real time, we used a molecular beacon assay with fluorescently labeled σ70 subunit of Escherichia coli RNAP. We show that substitutions and deletions in σ region 3.2 decrease the rate of promoter escape and lead to accumulation of inactive complexes during transcription initiation. Secondary channel factors differentially regulate this process depending on the promoter and mutations in σ region 3.2. GreA generally increase the rate of promoter escape; DksA also stimulates promoter escape on certain templates, while GreB either stimulates or inhibits this process depending on the template. When observed, the stimulation of promoter escape correlates with the accumulation of stressed transcription complexes with scrunched DNA, while changes in the RNA 5′-end structure modulate promoter clearance. Thus, the initiation-to-elongation transition is controlled by a complex interplay between RNAP-binding protein factors and the growing RNA chain.

High-Resolution Crystal Structure of RpoS Fragment Including a Partial Region 1.2 and Region 2 from the Intracellular Pathogen Legionella pneumophila

Legionella pneumophila RpoS (LpRpoS) is an alternative sigma factor of RNA polymerase (RNAP) essential for virulence and stress resistance. To investigate the mechanism of RpoS in the intracellular pathogen L. pneumophila, we determined the high-resolution crystal structure of the LpRpoS (residues 95-194) containing a partial region 1.2 and region 2. The structure of LpRpoS (residues 95-194) reveals that the conserved residues are critical for promoter melting, DNA and core RNAP binding. The differences in regulatory factor binding site between Escherichia coli RpoS and LpRpoS suggest that LpRpoS may employ a distinct mechanism to recruit alternative regulatory factors controlling transcription initiation.

Dual role of MsRbpA: transcription activation and rescue of transcription from the inhibitory effect of rifampicin

MsRbpA is an RNA polymerase (RNAP) binding protein from Mycobacterium smegmatis. According to previous studies, MsRbpA rescues rifampicin-induced transcription inhibition upon binding to the RNAP. Others have shown that RbpA from Mycobacterium tuberculosis (MtbRbpA) is a transcription activator. In this study, we report that both MsRbpA and MtbRbpA activate transcription as well as rescue rifampicin-induced transcription inhibition. Transcription activation is achieved through the increased formation of closed RNAP–promoter complex as well as enhanced rate of conversion of this complex to a stable transcriptionally competent RNAP–promoter complex. When a 16 aa peptide fragment (Asp 58 to Lys 73) was deleted from MsRbpA, the resulting protein showed 1000-fold reduced binding with core RNAP. The deletion results in abolition of transcription activation and rescue of transcription from the inhibitory effect of rifampicin. Through alanine scanning of this essential region of MsRbpA, Gly 67, Val 69, Pro 70 and Pro 72 residues are identified to be important for MsRbpA function. Furthermore, we report here that the protein is indispensable for M. smegmatis, and it appears to help the organism grow in the presence of the antibiotic rifampicin.

Transcriptional repressor CopR acts by inhibiting RNA polymerase binding

CopR is a transcriptional repressor encoded by the broad-host-range streptococcal plasmid pIP501, which also replicates in Bacillus subtilis. It acts in concert with the antisense RNA, RNAIII, to control pIP501 replication. CopR represses transcription of the essential repR mRNA about 10- to 20-fold. In previous work, DNA binding and dimerization constants were determined and the motifs responsible localized. The C terminus of CopR was shown to be required for stability. Furthermore, SELEX of the copR operator revealed that in vivo evolution was for maximal binding affinity. Here, we elucidate the repression mechanism of CopR. Competition assays showed that CopR–operator complexes are 18-fold less stable than RNA polymerase (RNAP)–pII complexes. DNase I footprinting revealed that the binding sites for CopR and RNAP overlap. Gel-shift assays demonstrated that CopR and B. subtilis RNAP cannot bind simultaneously, but compete for binding at promoter pII. Due to its higher intracellular concentration CopR inhibits RNAP binding. Additionally, KMnO4 footprinting experiments indicated that prevention of open complex formation at pII does not further contribute to the repression effect of CopR.

Evidence that Regulatory Protein MarA of Escherichia coli Represses rob by Steric Hindrance

ABSTRACT The MarA protein of Escherichia coli can both activate and repress the initiation of transcription, depending on the position and orientation of its degenerate 20-bp binding site (“marbox”) at the promoter. For all three known repressed genes, the marbox overlaps the promoter. It has been reported that MarA represses the rob promoter via an RNA polymerase (RNAP)-DNA-MarA ternary complex. Under similar conditions, we found a ternary complex for the repressed purA promoter also. These findings, together with the backwards orientation of repressed marboxes, suggested a unique interaction of MarA with RNAP in repression. However, no repression-specific residues of MarA could be found among 38 single-alanine replacement mutations previously shown to retain activation function or among mutants from random mutagenesis. Mutations Thr12Ala, Arg36Ala, Thr95Ile, and Pro106Ala were more damaging for activation than for repression, some up to 10-fold, so these residues may play a specific role in activation. We found that nonspecific binding of RNAP to promoterless regions of DNA was presumably responsible for the ternary complexes seen previously. When RNAP binding was promoter specific, MarA reduced RNAP access to the rob promoter; there was little or no ternary complex. These findings strongly implicate steric hindrance as the mechanism of repression of rob by MarA.

m-Xylene-Responsive Pu-PnifH Hybrid σ54 Promoters That Overcome Physiological Control in Pseudomonas putida KT2442

ABSTRACT The sequences surrounding the −12/−24 motif of the m-xylene-responsive σ54 promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH σ54 promoter variants known to have various degrees of efficacy and affinity for σ54-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XylR) fused to the −12/−24 region of the wild-type PnifH and its higher σ54-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XylR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the −12/−24 region of Pu dictates the coupling of promoter output to growth conditions.

Multiple Regions on the Escherichia coliHeat Shock Transcription Factor ς32 Determine Core RNA Polymerase Binding Specificity

ABSTRACT We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the rpoHgene, which encodes ς32 in Escherichia coli. All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2. Based on the estimatedKs s for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes. The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants’ effect on core RNAP interaction was only clearly seen in the presence of ς70 competitor. The class I mutant behaved nearly identically to the wild type in core RNAP binding. Two point mutations in class III altered residues that were distant from one another. One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors. These data suggest that there is more than one core RNAP binding region in ς32 and that differences in contact sites probably exist among sigma factors.