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• W2009783713 abstract "In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the β gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the β′ clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity. In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the β gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the β′ clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity. The β gate loop of bacterial RNA polymerase mediates antipausing by RfaH and NusG RfaH and NusG interact with the β gate loop and β′ clamp to bridge the DNA channel These bridging contacts may rigidify RNA polymerase to resist pausing and termination NusG homologs present in all organisms may employ similar processivity mechanisms The structure, effects on RNA synthesis, and the major binding site on the RNAP of proteins from the NusG family are universally conserved (Belogurov et al., 2009Belogurov G.A. Mooney R.A. Svetlov V. Landick R. Artsimovitch I. Functional specialization of transcription elongation factors.EMBO J. 2009; 28: 112-122Crossref PubMed Scopus (95) Google Scholar, Hirtreiter et al., 2010Hirtreiter A. Damsma G.E. Cheung A.C. Klose D. Grohmann D. Vojnic E. Martin A.C. Cramer P. Werner F. Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif.Nucleic Acids Res. 2010; 38: 4040-4051Crossref PubMed Scopus (125) Google Scholar, Mooney et al., 2009bMooney R.A. Schweimer K. Rösch P. Gottesman M. Landick R. Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators.J. Mol. Biol. 2009; 391: 341-358Crossref PubMed Scopus (144) Google Scholar). These proteins are also involved in crosstalk between transcription and other key cellular processes, such as RNA processing and translation (Bies-Etheve et al., 2009Bies-Etheve N. Pontier D. Lahmy S. Picart C. Vega D. Cooke R. Lagrange T. RNA-directed DNA methylation requires an AGO4-interacting member of the SPT5 elongation factor family.EMBO Rep. 2009; 10: 649-654Crossref PubMed Scopus (112) Google Scholar, Burmann et al., 2010Burmann B.M. Schweimer K. Luo X. Wahl M.C. Stitt B.L. Gottesman M.E. Rösch P. A NusE:NusG complex links transcription and translation.Science. 2010; 328: 501-504Crossref PubMed Scopus (241) Google Scholar, Peterlin and Price, 2006Peterlin B.M. Price D.H. Controlling the elongation phase of transcription with P-TEFb.Mol. Cell. 2006; 23: 297-305Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, Schneider et al., 2006Schneider D.A. French S.L. Osheim Y.N. Bailey A.O. Vu L. Dodd J. Yates J.R. Beyer A.L. Nomura M. RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing.Proc. Natl. Acad. Sci. USA. 2006; 103: 12707-12712Crossref PubMed Scopus (81) Google Scholar, Wang and Dennis, 2009Wang M.B. Dennis E.S. SPT5-like, a new component in plant RdDM.EMBO Rep. 2009; 10: 573-575Crossref PubMed Scopus (5) Google Scholar). The Escherichia coli NusG and RfaH are the best studied members of this family. NusG is a general transcription factor that is essential in the wild-type E. coli and is bound to nearly all transcribed genes (Mooney et al., 2009aMooney R.A. Davis S.E. Peters J.M. Rowland J.L. Ansari A.Z. Landick R. Regulator trafficking on bacterial transcription units in vivo.Mol. Cell. 2009; 33: 97-108Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). RfaH, a sequence-specific paralog of NusG, preferentially increases distal expression in operons containing promoter-proximal ops DNA elements. The ops sequence mediates RfaH binding to elongating RNAP and thus restricts RfaH action to just a few E. coli operons (Belogurov et al., 2009Belogurov G.A. Mooney R.A. Svetlov V. Landick R. Artsimovitch I. Functional specialization of transcription elongation factors.EMBO J. 2009; 28: 112-122Crossref PubMed Scopus (95) Google Scholar); consistently, rfaH is dispensable for cell viability. The N-terminal domains of RfaH and NusG are structurally homologous, are predicted to make similar contacts with RNAP, and are sufficient in vitro for the reduction of transcriptional pausing (Belogurov et al., 2009Belogurov G.A. Mooney R.A. Svetlov V. Landick R. Artsimovitch I. Functional specialization of transcription elongation factors.EMBO J. 2009; 28: 112-122Crossref PubMed Scopus (95) Google Scholar, Mooney et al., 2009bMooney R.A. Schweimer K. Rösch P. Gottesman M. Landick R. Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators.J. Mol. Biol. 2009; 391: 341-358Crossref PubMed Scopus (144) Google Scholar). However, full-length RfaH and NusG have opposite effects on the expression of poorly translated genes (e.g., foreign DNA) in vivo. NusG acts together with ρ to terminate transcription (Cardinale et al., 2008Cardinale C.J. Washburn R.S. Tadigotla V.R. Brown L.M. Gottesman M.E. Nudler E. Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli.Science. 2008; 320: 935-938Crossref PubMed Scopus (223) Google Scholar, Mooney et al., 2009bMooney R.A. Schweimer K. Rösch P. Gottesman M. Landick R. Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators.J. Mol. Biol. 2009; 391: 341-358Crossref PubMed Scopus (144) Google Scholar), whereas RfaH reduces termination via its antipausing (AP) activity and exclusion of NusG (Belogurov et al., 2009Belogurov G.A. Mooney R.A. Svetlov V. Landick R. Artsimovitch I. Functional specialization of transcription elongation factors.EMBO J. 2009; 28: 112-122Crossref PubMed Scopus (95) Google Scholar, Carter et al., 2004Carter H.D. Svetlov V. Artsimovitch I. Highly divergent RfaH orthologs from pathogenic proteobacteria can substitute for Escherichia coli RfaH both in vivo and in vitro.J. Bacteriol. 2004; 186: 2829-2840Crossref PubMed Scopus (27) Google Scholar). RfaH and other antiterminators, such as the phage λ N and Q proteins (Roberts et al., 2008Roberts J.W. Shankar S. Filter J.J. RNA polymerase elongation factors.Annu. Rev. Microbiol. 2008; 62: 211-233Crossref PubMed Scopus (85) Google Scholar), appear to maintain the elongating RNAP complex (EC) in a rapidly moving mode by preventing its isomerization into an off-pathway state called an “elemental pause” (Ederth et al., 2006Ederth J. Mooney R.A. Isaksson L.A. Landick R. Functional interplay between the jaw domain of bacterial RNA polymerase and allele-specific residues in the product RNA-binding pocket.J. Mol. Biol. 2006; 356: 1163-1179Crossref PubMed Scopus (29) Google Scholar). From this state, the paused EC is thought to isomerize into long-lived paused states (e.g., upon backtracking or formation of a nascent RNA hairpin) or to enter a termination pathway (Artsimovitch and Landick, 2000Artsimovitch I. Landick R. Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals.Proc. Natl. Acad. Sci. USA. 2000; 97: 7090-7095Crossref PubMed Scopus (321) Google Scholar). Formation of the elemental pause probably involves fraying of the 3′ RNA nucleotide and rearrangement of an active-site element called the trigger loop and occurs after nucleotide addition, but before RNAP translocation (Sydow et al., 2009Sydow J.F. Brueckner F. Cheung A.C. Damsma G.E. Dengl S. Lehmann E. Vassylyev D. Cramer P. Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA.Mol. Cell. 2009; 34: 710-721Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, Toulokhonov et al., 2007Toulokhonov I. Zhang J. Palangat M. Landick R. A central role of the RNA polymerase trigger loop in active-site rearrangement during transcriptional pausing.Mol. Cell. 2007; 27: 406-419Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Thus, a regulator could suppress pausing either by promoting translocation or by preventing active-site conformational changes, but the molecular mechanism of an AP modification remains unknown for any protein. RfaH is an excellent model to address the question of how a regulator can modify RNAP to resist pause signals. RfaH action can be readily monitored in the cell because it is dispensable and controls just a few operons. In addition, the RfaH structure, mechanism of recruitment, DNA- and RNAP-binding regions, and binding site on the RNAP have been determined. RfaH binds to the β′ clamp helices domain (β′CH) and the nontemplate (NT) DNA strand at the upstream end of the transcription bubble (Belogurov et al., 2007Belogurov G.A. Vassylyeva M.N. Svetlov V. Klyuyev S. Grishin N.V. Vassylyev D.G. Artsimovitch I. Structural basis for converting a general transcription factor into an operon-specific virulence regulator.Mol. Cell. 2007; 26: 117-129Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Unlike the phage antiterminators that also stabilize the EC against dissociation (Rees et al., 1997Rees W.A. Weitzel S.E. Das A. von Hippel P.H. Regulation of the elongation-termination decision at intrinsic terminators by antitermination protein N of phage lambda.J. Mol. Biol. 1997; 273: 797-813Crossref PubMed Scopus (49) Google Scholar, Yarnell and Roberts, 1999Yarnell W.S. Roberts J.W. Mechanism of intrinsic transcription termination and antitermination.Science. 1999; 284: 611-615Crossref PubMed Scopus (264) Google Scholar), RfaH does not affect the EC stability or intrinsic termination, except at one unusual signal (Artsimovitch and Landick, 2002Artsimovitch I. Landick R. The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand.Cell. 2002; 109: 193-203Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, Carter et al., 2004Carter H.D. Svetlov V. Artsimovitch I. Highly divergent RfaH orthologs from pathogenic proteobacteria can substitute for Escherichia coli RfaH both in vivo and in vitro.J. Bacteriol. 2004; 186: 2829-2840Crossref PubMed Scopus (27) Google Scholar), allowing direct testing of its AP mechanism. In the simplest scenario, RfaH may favor reannealing of the DNA strands and thus push the RNAP forward; indeed, we showed that RfaH favors forward translocation of the enzyme (Svetlov et al., 2007Svetlov V. Belogurov G.A. Shabrova E. Vassylyev D.G. Artsimovitch I. Allosteric control of the RNA polymerase by the elongation factor RfaH.Nucleic Acids Res. 2007; 35: 5694-5705Crossref PubMed Scopus (59) Google Scholar). However, the location of the RfaH binding site on the EC suggests that the bound RfaH may additionally restrict movements of the β′ clamp that have been proposed to accompany pausing (Landick, 2001Landick R. RNA polymerase clamps down.Cell. 2001; 105: 567-570Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Here we present the evidence in support of the second mechanism. We show that RfaH action depends on two RNAP elements, the β′CH and the β subunit gate loop (βGL), which are located directly across from each other on the two sides on the DNA-binding channel. We propose that β′CH, βGL, and RfaH jointly form a clamp on the DNA that allows the RNAP to resist pausing and termination signals. To identify the determinants required for AP by RfaH, we previously carried out mutational analysis of the N-terminal domain, RfaHN, which is sufficient for all in vitro activities of RfaH. This analysis (Belogurov et al., 2010Belogurov G.A. Sevostyanova A. Svetlov V. Artsimovitch I. Functional regions of the N-terminal domain of the antiterminator RfaH.Mol. Microbiol. 2010; 76: 286-301Crossref PubMed Scopus (51) Google Scholar) revealed three functional regions (Figure 1A ): (1) a cluster of residues that probably interact with the NT DNA, (2) a hydrophobic surface that is predicted to make van der Waals contacts with the β′CH, and (iii) a cluster of three residues (HTT) distant from the NT DNA and the β′CH. Substitutions in the first two regions are expected to compromise RfaH interactions with the EC; indeed, the defects of these substitutions are suppressed at high RfaH concentrations. In contrast, RfaH variants with substitutions of the HTT residues display dramatically reduced AP activity even when present at high concentrations (Belogurov et al., 2010Belogurov G.A. Sevostyanova A. Svetlov V. Artsimovitch I. Functional regions of the N-terminal domain of the antiterminator RfaH.Mol. Microbiol. 2010; 76: 286-301Crossref PubMed Scopus (51) Google Scholar). These observations are inconsistent with a model in which RfaH binding to the DNA and the β′CH is sufficient for its AP activity and suggest that an additional interaction between the HTT motif and the EC is required. The goal of this study was to characterize this interaction. First, we wanted to exclude a possibility that even though the HTT residues are not modeled to interact with either the DNA or the β′CH (Figure 1B), their substitutions compromise RfaH binding to the EC indirectly and an altered protein dissociates after recruitment. To ascertain that RfaH variants with HTT residues substituted for Ala remain bound to the EC, we used a σ competition assay that relies on the RfaH ability to sterically block σ recruitment to the EC, because as we showed earlier, both proteins bind to the β′CH domain (Sevostyanova et al., 2008Sevostyanova A. Svetlov V. Vassylyev D.G. Artsimovitch I. The elongation factor RfaH and the initiation factor sigma bind to the same site on the transcription elongation complex.Proc. Natl. Acad. Sci. USA. 2008; 105: 865-870Crossref PubMed Scopus (55) Google Scholar). To test the ability of altered RfaH variants to compete with σ during elongation, we carried out a standard single-round transcription assay (Figure 1C) by using a linear DNA template with a strong T7A1 promoter followed by an initial transcribed region that allows for formation of radiolabeled ECs stalled after incorporation of a G residue at position 37 (G37) when transcription is initiated in the absence of UTP. The template also encodes the ops pause signal (opsP), which induces RNAP backtracking and mediates RfaH recruitment to the EC, the consensus −10 element, and a hairpin-stabilized his pause signal (hisP). Upon addition of all NTPs, rifapentin (to block reinitiation), and the excess of free σ (1 μM versus 30 nM EC), RNAP elongated the nascent RNA, transiently pausing at the opsP (U43) and the hisP sites; σ contacts to bases in a −10-like sequence induced a very long pause at the σP site from which RNAP did not escape under the conditions of our experiments (Figure 1C). The wild-type (WT) RfaH, when present, delayed RNAP escape from the C45 position (through interactions with the ops bases) and accelerated transcription at other sites, as evidenced by a faster RNAP arrival at the end of the template (RO). However, on this template, AP effects of RfaH are difficult to quantify because the hisP signal, which has been shown to respond to RfaH (Artsimovitch and Landick, 2002Artsimovitch I. Landick R. The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand.Cell. 2002; 109: 193-203Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), is separated from the halted complex position by ∼100 nucleotides encoding two additional strong and many weak pause sites, which desynchronize the RNAP population. We have reported the effects of all RfaH variants on pausing at hisP on a less complex template (Belogurov et al., 2010Belogurov G.A. Sevostyanova A. Svetlov V. Artsimovitch I. Functional regions of the N-terminal domain of the antiterminator RfaH.Mol. Microbiol. 2010; 76: 286-301Crossref PubMed Scopus (51) Google Scholar). Most importantly, the wild-type RfaH strongly inhibited pausing at the σP site even at a 15-fold molar excess of σ. We found that H65A and T67A (and T66A, not shown) variants also competed with σ efficiently, indicating that the inability of these variants to accelerate elongation is not due to their failure to remain bound to the EC after recruitment. As a control, we used an RfaH Y54F variant with a substitution at the RfaH/β′CH interface (Figure 1B); this substitution confers a dramatic AP defect that can be rescued at 3 μM RfaH (Belogurov et al., 2010Belogurov G.A. Sevostyanova A. Svetlov V. Artsimovitch I. Functional regions of the N-terminal domain of the antiterminator RfaH.Mol. Microbiol. 2010; 76: 286-301Crossref PubMed Scopus (51) Google Scholar). Y54F was initially recruited at the ops site (as reflected by reduced pausing at U43), but apparently failed to maintain stable postrecruitment interactions: it neither accelerated elongation nor competed with σ (Figure 1C). These results suggest that the HTT residues are largely dispensable for RfaH binding to the EC, but may be involved in a functional interaction required for the AP modification. The model of EC bound to RfaH (Belogurov et al., 2007Belogurov G.A. Vassylyeva M.N. Svetlov V. Klyuyev S. Grishin N.V. Vassylyev D.G. Artsimovitch I. Structural basis for converting a general transcription factor into an operon-specific virulence regulator.Mol. Cell. 2007; 26: 117-129Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) positions the HTT motif near the βGL (Figure 1B). To evaluate a possible role of the E. coli βGL in RfaH function, we substituted the β residues 368–376 with two glycines. Surprisingly, although the βGL has been proposed to play a key role in DNA loading during initiation (Vassylyev et al., 2002Vassylyev D.G. Sekine S. Laptenko O. Lee J. Vassylyeva M.N. Borukhov S. Yokoyama S. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution.Nature. 2002; 417: 712-719Crossref PubMed Scopus (627) Google Scholar), we found that the ΔβGL variant was active in vitro (Figure 2) and under certain conditions in vivo (Figure S1, available online). We first tested whether the deletion of the βGL would abolish the RNAP response to RfaH in vitro at the hisP signal. In these experiments, we used the isolated RfaHN, which does not require an ops element for recruitment (Belogurov et al., 2007Belogurov G.A. Vassylyeva M.N. Svetlov V. Klyuyev S. Grishin N.V. Vassylyev D.G. Artsimovitch I. Structural basis for converting a general transcription factor into an operon-specific virulence regulator.Mol. Cell. 2007; 26: 117-129Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), thereby simplifying the kinetic analysis. In a single-round pause assay, RfaHN accelerated RNAP escape from hisP almost 4-fold, reducing the pause half-life from 41 to 11 s, but had almost no effect on the ΔβGL enzyme (Figure 2A). This result shows that the βGL is necessary for the AP activity of RfaH, at least at one model pause site. To test whether the βGL deletion confers a general AP defect (as opposed to an effect specific to the his pause), we compared elongation of an rpoB mRNA, which is devoid of strong regulatory pauses, but punctuated by many elemental pauses that contribute at least 65% to the overall rate of elongation (Neuman et al., 2003Neuman K.C. Abbondanzieri E.A. Landick R. Gelles J. Block S.M. Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking.Cell. 2003; 115: 437-447Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar), by using the deletion and the wild-type enzymes. RfaHN increased the apparent elongation rate by the wild-type RNAP by 1.8-fold, but slowed down the deletion enzyme ∼1.4-fold (Figure 2B); RfaHN has a similar inhibitory effect on the wild-type enzyme at higher NTPs (Svetlov et al., 2007Svetlov V. Belogurov G.A. Shabrova E. Vassylyev D.G. Artsimovitch I. Allosteric control of the RNA polymerase by the elongation factor RfaH.Nucleic Acids Res. 2007; 35: 5694-5705Crossref PubMed Scopus (59) Google Scholar). One explanation for this phenotype could be that the βGL deletion renders RNAP pause resistant. We reported previously that RfaH does not accelerate transcription at saturating substrate concentrations or by “fast” RNAP variants with substitutions in β and β′ subunits that render the enzyme insensitive to pause and termination signals (Svetlov et al., 2007Svetlov V. Belogurov G.A. Shabrova E. Vassylyev D.G. Artsimovitch I. Allosteric control of the RNA polymerase by the elongation factor RfaH.Nucleic Acids Res. 2007; 35: 5694-5705Crossref PubMed Scopus (59) Google Scholar). In particular, an enzyme that is missing a large sequence insertion in the β′ trigger loop, Δβ′SI3 RNAP, fails to respond to RfaH. However, the ΔβGL RNAP was only moderately faster than the wild-type RNAP and displayed a dramatically different response to the hisP signal in the presence and in the absence of RfaH in a side-by-side comparison with the Δβ′SI3 enzyme (Figure 2A). If the βGL deletion removes a contact with the HTT motif, substitutions of the HTT residues, which abolish RfaH effects on transcription by the wild-type RNAP, should not have any additional effects on the ΔβGL enzyme. Indeed, we found that the in vitro transcription pattern of the ΔβGL enzyme was the same with the wild-type, H65A, T66A, and T67A RfaH variants (Figure S2). Together, these results demonstrate that the βGL is required in vitro for the AP effects of RfaH and are consistent with a functional interaction between βGL and HTT. To ascertain that the βGL deletion does not eliminate RfaH binding to the EC, we used σ competition and electrophoretic mobility shift assays (Figure 3). We found that RfaHN, which does not dissociate from the EC in vitro and thus mimics an in vivo pattern (Belogurov et al., 2007Belogurov G.A. Vassylyeva M.N. Svetlov V. Klyuyev S. Grishin N.V. Vassylyev D.G. Artsimovitch I. Structural basis for converting a general transcription factor into an operon-specific virulence regulator.Mol. Cell. 2007; 26: 117-129Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), eliminated σ recruitment to both the wild-type and the ΔβGL ECs (Figure 3A). As expected, RfaHN drastically altered the pattern of transcription by the wild-type RNAP, delaying escape from the ops site and reducing pausing at the downstream signals, such as hisP. In contrast, RfaHN had no effect on the ΔβGL RNAP, except at the σP site where it abolished pausing as efficiently as with the wild-type RNAP. The full-length RfaH had similar effects (data not shown). We next visualized RfaH binding to the ops-paused ECs by a mobility shift assay (Figure 3B). We assembled ECs on nucleic acid scaffolds composed of the template and NT DNA strands and the RNA (see Experimental Procedures); we used this assay previously to identify the RfaH target on the EC (Artsimovitch and Landick, 2002Artsimovitch I. Landick R. The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand.Cell. 2002; 109: 193-203Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). To verify that the assembled complexes were active, we incubated unlabeled ECs with radiolabeled α[32P]GTP, the incoming nucleotide specified by the template DNA. When we incorporated α[32P]GMP, the position of the ECs could be readily visualized in an agarose gel (Figure 3B, left). To monitor RfaH binding to the EC, we radiolabeled RfaH via a kinase-recognition sequence at the N terminus of the protein. RfaH did not enter the gel when present alone, but comigrated with ECs assembled with the wild-type and ΔβGL RNAPs (Figure 3B, right). By contrast, a β′I290R substitution at the tip of the β′CH completely abolished RfaH binding (but not GMP incorporation), consistent with a model in which the β′CH is the major affinity determinant for RfaH. Together, these results argue that the βGL is dispensable for RfaH binding. Consistently, we detected only a very weak (but apparently specific) interaction between RfaHN and the β N-terminal domain in a bacterial two-hybrid assay (Figure S3). Many RfaH-controlled genes are horizontally transferred (Belogurov et al., 2009Belogurov G.A. Mooney R.A. Svetlov V. Landick R. Artsimovitch I. Functional specialization of transcription elongation factors.EMBO J. 2009; 28: 112-122Crossref PubMed Scopus (95) Google Scholar) and are thus expected to be subject to polarity control by ρ (Cardinale et al., 2008Cardinale C.J. Washburn R.S. Tadigotla V.R. Brown L.M. Gottesman M.E. Nudler E. Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli.Science. 2008; 320: 935-938Crossref PubMed Scopus (223) Google Scholar). RfaH has been reported to inhibit the action of ρ in vitro (Belogurov et al., 2009Belogurov G.A. Mooney R.A. Svetlov V. Landick R. Artsimovitch I. Functional specialization of transcription elongation factors.EMBO J. 2009; 28: 112-122Crossref PubMed Scopus (95) Google Scholar) and in vivo (Stevens et al., 1997Stevens M.P. Clarke B.R. Roberts I.S. Regulation of the Escherichia coli K5 capsule gene cluster by transcription antitermination.Mol. Microbiol. 1997; 24: 1001-1012Crossref PubMed Scopus (73) Google Scholar). We examined the effect of RfaH on the extent of termination (i.e., polarity) across the 11-gene rfb operon (rfbBDACX-glf-rfc-wbbIJKL; Figure 4A ). By using chromatin immunoprecipitation followed by tiling microarray analysis (ChIP-chip), we have shown that after recruitment at a promoter-proximal ops site (located 76 bp upstream of the first ORF, rfbB), RfaH remains bound throughout the entire rfb operon and excludes NusG from this and other ops-containing operons (Belogurov et al., 2009Belogurov G.A. Mooney R.A. Svetlov V. Landick R. Artsimovitch I. Functional specialization of transcription elongation factors.EMBO J. 2009; 28: 112-122Crossref PubMed Scopus (95) Google Scholar). At the same time, we detected significant association of ρ with the distal part of rfb operon (Mooney et al., 2009aMooney R.A. Davis S.E. Peters J.M. Rowland J.L. Ansari A.Z. Landick R. Regulator trafficking on bacterial transcription units in vivo.Mol. Cell. 2009; 33: 97-108Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), consistent with a notion that NusG is required for efficient RNA release by ρ, but not for ρ recruitment. To test the effects of RfaH and ρ on the rfb expression, we compared expression levels of the first (rfbB; shown in red in Figure 4) and the eighth (wbbI; blue) genes by qRT-PCR as described in Experimental Procedures. We observed strong polarity (defined here as the ratio of rfbB and wbbI messages) in the ΔrfaH strain, where wbbI mRNA was barely detectable (rfbB/wbbI ∼800; Figure 4B, right). Consistent with our expectations, essentially all polarity was caused by ρ, as polarity was eliminated upon the addition of the ρ inhibitor bicyclomycin (BCM; shaded bars in Figure 4B). In the absence of RfaH, addition of BCM increased the absolute level of wbbI RNA more than 700-fold. In the presence of RfaH, ∼45% of RNAP molecules reached wbbI gene (Figure 4B, left); this fraction increased to 78% when ρ was inhibited by BCM. These results show that even though RfaH does not completely abolish ρ-dependent termination (e.g., if not all ECs became modified by RfaH), it eliminates most of the ρ-mediated polarity within rfb operon. The dramatic effect of RfaH on ρ termination is mediated by two mechanisms. First, RfaH modifies RNAP into a pause-resistant state (Svetlov et al., 2007Svetlov V. Belogurov G.A. Shabrova E. Vassylyev D.G. Artsimovitch I. Allosteric control of the RNA polymerase by the elongation factor RfaH.Nucleic Acids Res. 2007; 35: 5694-5705Crossref PubMed Scopus (59) Google Scholar), thus making it less susceptible to ρ. Second, RfaH excludes the ρ stimulatory factor NusG through competition for binding to the β′CH. Thus, the levels of NusG associated with the rfb operon are very low (Figure 4A), despite the fact that NusG concentration in the cell far exceeds that of RfaH (Belogurov et al., 2009Belogurov G.A. Mooney R.A. Svetlov V. Landick R. Artsimovitch I. Functional specialization of transcription elongation factors.EMBO J. 2009; 28: 112-122Crossref PubMed Scopus (95) Google Scholar). In the absen" @default.
• W2009783713 created "2016-06-24" @default.
• W2009783713 creator A5010325555 @default.
• W2009783713 creator A5063187030 @default.
• W2009783713 creator A5070653259 @default.
• W2009783713 creator A5074407016 @default.
• W2009783713 creator A5076402263 @default.
• W2009783713 date "2011-07-01" @default.
• W2009783713 modified "2023-10-12" @default.
• W2009783713 title "The β Subunit Gate Loop Is Required for RNA Polymerase Modification by RfaH and NusG" @default.
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