Structural studies of antibiotics not only provide a short cut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become ‘frozen’ after inhibitor binding1,2. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism3. Here we report the structure of dMyx—a desmethyl derivative of myxopyronin B4—complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble5,6. Notably, binding of dMyx stabilizes refolding of the β’-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex—the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.

During transcription initiation by bacterial RNA polymerase, the sigma subunit recognizes the -35 and -10 promoter elements; free sigma, however, does not bind DNA. We selected ssDNA aptamers that strongly and specifically bound free sigma(A) from Thermus aquaticus. A consensus sequence, GTA(C/T)AATGGGA, was required for aptamer binding to sigma(A), with the TA(C/T)AAT segment making interactions similar to those made by the -10 promoter element (consensus sequence TATAAT) in the context of RNA polymerase holoenzyme. When in dsDNA form, the aptamers function as strong promoters for the T. aquaticus RNA polymerase sigma(A) holoenzyme. Recognition of the aptamer-based promoters depends on the downstream GGGA motif from the aptamers' common sequence, which is contacted by sigma(A) region 1.2 and directs transcription initiation even in the absence of the -35 promoter element. Thus, recognition of bacterial promoters is controlled by independent interactions of sigma with multiple basal promoter elements.

RNA polymerase is a target for numerous regulatory events in all living cells. Recent studies identified a few ''hot spots'' on the surface of bacterial RNA polymerase that mediate its interactions with diverse accessory proteins. Prominent among these hot spots, the ␤ subunit clamp helices serve as a major binding site for the initiation factor and for the elongation factor RfaH. Furthermore, the two proteins interact with the nontemplate DNA strand in transcription complexes and thus may interfere with each other's activity. We show that RfaH does not inhibit transcription initiation but, once recruited to RNA polymerase, abolishes -dependent pausing. We argue that this apparent competition is due to a steric exclusion of by RfaH that is stably bound to the nontemplate DNA and clamp helices, both of which are necessary for the recruitment to the transcription complex. Our findings highlight the key regulatory role played by the clamp helices during both initiation and elongation stages of transcription.clamp helices ͉ RNA polymerase ͉ transcription factor ͉ nontemplate DNA B acterial RNA polymerase (RNAP) is a principal target for numerous accessory proteins and small ligands that finetune gene expression profiles to match the cell needs. Competition (or cooperation) among these regulators for the finite number of targets on the RNAP surface determines the patterns of gene expression. The classical paradigm for the partitioning of the regulatory space is competition (1) with different initiation factors competing for binding to the core enzyme (subunit composition ␣ 2 ␤␤Ј ) and, when successful, directing it to a subset of -specific promoters. The -subunit makes many contacts to the core RNAP among which the ␤Ј subunit clamp helices (␤Ј CH, a coiled-coil motif comprising residues 260-309 in the Escherichia coli enzyme) are thought to constitute the major binding site in the free RNAP (2, 3) as well as in the transcription elongation complex (TEC) (4). Our recent finding that the ␤Ј CH is also required for recruitment of the elongation factor RfaH (5) suggested that competition for this site may regulate gene expression far beyond -specific promoter recognition.RfaH reduces pausing and termination thereby suppressing transcriptional polarity in long operons encoding virulence and fertility determinants (6, 7). RfaH action depends on the ops DNA sequence (GGCGGTAGnnTG) elements located in the transcribed regions of RfaH-controlled operons (7). In vitro, the ops element indeed mediates RfaH binding to the TEC but only if it is placed in the nontemplate (NT) DNA strand exposed on the surface of RNAP (7). RfaH recruitment is thought to occur in two steps: (i) sequence-specific binding of the N-terminal domain to DNA triggers displacement of the stably bound C-terminal domain to expose the RNAP binding site on the N-domain, and (ii) interactions of the N-domain with ␤Ј CH on one side and (nonspecific) interactions with the NT strand on the other allow for the stable retention of RfaH on the TEC throughout elongation...

Bacterial transcription factors DksA and GreB belong to a family of coiled-coil proteins that bind within the secondarychannel of RNA polymerase (RNAP). These proteins display structural homology but play different regulatory roles. DksA disrupts RNAP interactions with promoter DNA and inhibits formation of initiation complexes, sensitizing rRNA synthesis to changes in concentrations of ppGpp and NTPs. Gre proteins remodel the RNAP active site and facilitate cleavage of the nascent RNA in elongation complexes. However, DksA and GreB were shown to have overlapping effects during initiation, and in vivo studies suggested that DksA may also function at post-initiation steps. Here we show that DksA has many features of an elongation factor: it inhibits both RNA chain extension and RNA shortening by exonucleolytic cleavage or pyrophosphorolysis and increases intrinsic termination in vitro and in vivo. However, DksA has no effect on Rho- or Mfd-mediated RNA release or nascent RNA cleavage in backtracked complexes, the regulatory target of Gre factors. Our results reveal that DksA effects on elongating RNAP are very different from those of GreB, suggesting that these regulators recognize distinct states of the transcription complex.

Transcription elongation factors from the NusG family are ubiquitous from bacteria to humans and play diverse roles in the regulation of gene expression. These proteins consist of at least two domains. The N-terminal domains directly bind to the largest, β′ in bacteria, subunit of RNA polymerase (RNAP), whereas the C-terminal domains interact with other cellular components and serve as platforms for the assembly of large nucleoprotein complexes. Escherichia coli NusG and its paralog RfaH modify RNAP into a fast, pause-resistant state but the detailed molecular mechanism of this modification remains unclear since no high-resolution structural data are available for the E. coli system. We wanted to investigate whether Thermus thermophilus (Tth) NusG can be used as a model for structural studies of this family of regulators. Here, we show that Tth NusG slows down rather than facilitates transcript elongation by its cognate RNAP. On the other hand, similarly to the E. coli regulators, Tth NusG apparently binds near the upstream end of the transcription bubble, competes with σA, and favors forward translocation by RNAP. Our data suggest that the mechanism of NusG recruitment to RNAP is universally conserved even though the regulatory outcomes among its homologs may appear distinct.

SUMMARYRegulation of gene expression of lytic bacteriophage φYS40 that infects thermophilic bacterium Thermus thermophilus was investigated and three temporal classes of phage genes --early, middle, and late --were revealed. φYS40 does not encode a DNA-dependent RNA polymerase (RNAP) and must rely on host RNAP for transcription of its genes. Bioinformatic analysis using a model of Thermus promoters predicted 43 putative σ A -dependent −10/-35 class phage promoters. A randomly chosen subset of those promoters was shown to be functional in vivo and in vitro and to belong to the early temporal class. Macroarray analysis, primer extension, and bioinformatic predictions identified 36 viral middle and late promoters. These promoters have a single common consensus element, which resembles host σ A RNAP holoenzyme −10 promoter consensus element sequence. The mechanism responsible for the temporal control of the three classes of promoters remains unknown, since host σ A RNAP holoenzyme-purified from either infected or uninfected cells efficiently transcribed all φYS40 promoters in vitro. Interestingly, our data showed that during infection, there is a significant increase and decrease, respectively, of transcript amounts of host translation initiation factors IF2 and IF3. This finding, together with the fact that most middle and late φYS40 transcripts were found to be leaderless, suggests that the shift to late viral gene expression may also occur at the level of mRNA translation.

Interactions of RNA polymerase (RNAP) with nucleic acids must be tightly controlled to ensure precise and processive RNA synthesis. The RNAP β′-subunit Switch-2 (SW2) region is part of a protein network that connects the clamp domain with the RNAP body and mediates opening and closing of the active center cleft. SW2 interacts with the template DNA near the RNAP active center and is a target for antibiotics that block DNA melting during initiation. Here, we show that substitutions of a conserved Arg339 residue in the Escherichia coli RNAP SW2 confer diverse effects on transcription that include defects in DNA melting in promoter complexes, decreased stability of RNAP/promoter complexes, increased apparent KM for initiating nucleotide substrates (2- to 13-fold for different substitutions), decreased efficiency of promoter escape, and decreased stability of elongation complexes. We propose that interactions of Arg339 with DNA directly stabilize transcription complexes to promote stable closure of the clamp domain around nucleic acids. During initiation, SW2 may cooperate with the σ3.2 region to stabilize the template DNA strand in the RNAP active site. Together, our data suggest that SW2 may serve as a key regulatory element that affects transcription initiation and RNAP processivity through controlling RNAP/DNA template interactions.

Bacterial RNA polymerase holoenzyme relies on its subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the subunit are positioned at an optimal distance to allow specific recognition of the ؊10 and ؊35 promoter elements, respectively. In free , the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with region 1 playing a role in the masking. To analyze the DNA-binding properties of the free , we selected single-stranded DNA aptamers that are specific to primary subunits from several bacterial species, including Escherichia coli and Thermus aquaticus. The aptamers share a consensus motif, TGTAGAAT, that is similar to the extended ؊10 promoter. We demonstrate that recognition of this motif by region 2 occurs without major structural rearrangements of observed upon the holoenzyme formation and is not inhibited by regions 1 and 4. Thus, the complex process of the ؊10 element recognition by RNA polymerase holoenzyme can be reduced to a simple system consisting of an isolated subunit and a short aptamer oligonucleotide. Recognition of the Ϫ10 element can be modeled in a complex of holo-RNAP with short oligonucleotides corresponding to the nontemplate promoter strand and containing the Ϫ10 sequence (nontemplate oligonucleotides) (6 -9). However, despite playing a crucial role in promoter recognition by holo-RNAP, free is unable to specifically recognize either promoters or nontemplate oligonucleotides (1, 10 -12).Crystal structures of holo-RNAPs from Thermus aquaticus and Thermus thermophilus revealed that contains three domains (2, 3, and 4, named after corresponding conserved regions) connected by flexible linkers (13,14). In holoenzyme, these domains are spread on the core enzyme surface, with 2 and 4 being positioned at an optimal distance relative to each other to allow interactions with the Ϫ10 and Ϫ35 promoter elements. The structure of the isolated individual domains of is almost identical to their structures in holo-RNAP (13-16). However, the structure of the full-length subunit in a free state remains unknown.Based on indirect biochemical and biophysical data, several mechanisms of inhibition of DNA-binding activity in free have been proposed. First, the N-terminal region of (region 1.1) was shown to inhibit DNA binding. Deletion of this region allows 70 to weakly bind double-stranded promoter DNA with some specificity (17, 18) but does not allow the recognition of single-stranded nontemplate oligonucleotides by free (15,19). It was proposed that region 1.1 may inhibit DNA binding by (i) direct masking of region 4 (17, 18), (ii) direct interactions with region 2 (20, 21), or (iii) by an indirect allosteric mechanism (22).Second, it was shown that free adopts a compact conformation in which DNA-recognition domains 2 and 4 are brought closer to each other than in the holoenzyme (23). The sub-optimal positioning of 2 and 4 is likely to interfere with simultaneous recognition of the Ϫ10 and Ϫ35 elements. Furthermore, the ...