RNA Polymerase Conformation Research Articles

Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response σB factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that σB is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results reveal that a non-conserved region of σ is an allosteric controller of transcription initiation and demonstrate how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation.

Structural basis for transcription complex disruption by the Mfd translocase.

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (ECs). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.

E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation.

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

Transcription inhibition by the depsipeptide antibiotic salinamide A.

We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center ‘bridge-helix cap’ comprising the ‘bridge-helix N-terminal hinge’, ‘F-loop’, and ‘link region’. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.DOI: http://dx.doi.org/10.7554/eLife.02451.001

Antisense Oligonucleotide-stimulated Transcriptional Pausing Reveals RNA Exit Channel Specificity of RNA Polymerase and Mechanistic Contributions of NusA and RfaH

Transcript elongation by bacterial RNA polymerase (RNAP) is thought to be regulated at pause sites by open versus closed positions of the RNAP clamp domain, pause-suppressing regulators like NusG and RfaH that stabilize the closed-clampRNAP conformation, and pause-enhancing regulators like NusA and exit channel nascent RNA structures that stabilize the open clamp RNAP conformation. However, the mutual effects of these protein and RNA regulators on RNAP conformation are incompletely understood. For example, it is unknown whether NusA directly interacts with exit channel duplexes and whether formation of exit channel duplexes and RfaH binding compete by favoring the open and closed RNAP conformations. We report new insights into these mechanisms using antisense oligonucleotide mimics of a pause RNA hairpin from the leader region of the his biosynthetic operon of enteric bacteria like Escherichia coli. By systematically varying the structure and length of the oligonucleotide mimic, we determined that full pause stabilization requires an RNA-RNA duplex of at least 8 bp or a DNA-RNA duplex of at least 11 bp; RNA-RNA duplexes were more effective than DNA-RNA. NusA stimulation of pausing was optimal with 10-bp RNA-RNA duplexes and was aided by single-stranded RNA upstream of the duplex but was significantly reduced with DNA-RNA duplexes. Our results favor direct NusA stabilization of exit channel duplexes, which consequently affect RNAP clamp conformation. Effects of RfaH, which suppresses oligo-stabilization of pausing, were competitive with antisense oligonucleotide concentration, suggesting that RfaH and exit channel duplexes compete via opposing effects on RNAP clamp conformation.

Cys-Pair Reporters Detect a Constrained Trigger Loop in a Paused RNA Polymerase

Transcriptional pausing, which regulates transcript elongation in both prokaryotes and eukaryotes, is thought to involve formation of alternative RNA polymerase conformations in which nucleotide addition is inhibited in part by restriction of trigger loop (TL) folding. The polymorphous TL must convert from a random coil to a helical hairpin that contacts the nucleotide triphosphate (NTP) substrate to allow rapid nucleotide addition. Understanding the distribution of TL conformations in different enzyme states is made difficult by the TL's small size and sensitive energetics. Here, we report a Cys-pair reporter strategy to elucidate the relative occupancies of different TL conformations in E. coli RNA polymerase based on the ability of Cys residues engineered into the TL and surrounding regions to form disulfide bonds. Our results indicate that a paused complex stabilized by a nascent RNA hairpin favors nonproductive TL conformations that persist after NTP binding but can be reversed by the elongation factor RfaH.

The Role of Bacterial Enhancer Binding Proteins as Specialized Activators of σ54-Dependent Transcription

Bacterial enhancer binding proteins (bEBPs) are transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ(54). We present a comprehensive and detailed summary of recent advances in our understanding of how these specialized molecular machines function. The review is structured by introducing each of the three domains in turn: the central catalytic domain, the N-terminal regulatory domain, and the C-terminal DNA binding domain. The role of the central catalytic domain is presented with particular reference to (i) oligomerization, (ii) ATP hydrolysis, and (iii) the key GAFTGA motif that contacts σ(54) for remodeling. Each of these functions forms a potential target of the signal-sensing N-terminal regulatory domain, which can act either positively or negatively to control the activation of σ(54)-dependent transcription. Finally, we focus on the DNA binding function of the C-terminal domain and the enhancer sites to which it binds. Particular attention is paid to the importance of σ(54) to the bacterial cell and its unique role in regulating transcription.

Use of Fast DNA Footprinting and Real-Time Fluorescence Spectroscopy to Characterize Novel, Biologically Revlevant Transcription Initiation Intermediates for E. Coli RNA Polymerase

RNA polymerase (RNAP) is a complex molecular motor responsible for catalyzing the synthesis of RNA from DNA in a promoter sequence-dependent manner. Knowledge of the mechanism of transcription initiation is crucial to understanding the regulation of gene expression. However, the RNAP-DNA intermediates on the pathway to open complex formation during transcription initiation are extremely short-lived and thus have been very difficult to characterize using traditional structural methods. Here, we present work focused on developing rapid, solution-based methods that allow us to characterize RNAP-DNA structures in the millisecond time regime. These include: fast DNA footprinting (using hydroxyl radical and permanganate probes) to characterize the interaction of RNAP with the DNA backbone and the extent of thymine base unstacking; stopped-flow FRET experiments to monitor DNA bending in real time; and 2-aminopurine fluorescence experiments to characterize the kinetics of DNA opening. In a related project, we utilize thin layer chromatography and fluorescence spectroscopy to characterize the short (2, 3 and 4-mer) RNA products made by RNAP during repeated rounds of abortive cycling before the enzyme progresses to forming full-length RNA transcripts. These combinatorial approaches have allowed us to determine the timing of loading of duplex DNA into the RNAP active site cleft and the kinetic mechanism of large-scale RNAP conformation changes leading to initial RNA synthesis. These research activities have successfully developed new methods to characterize transient intermediates in solution and to begin to develop a detailed structural-mechanistic picture of transcription initiation.

Nitric oxide-responsive interdomain regulation targets the σ54-interaction surface in the enhancer binding protein NorR

Bacterial enhancer binding proteins (bEBPs) are specialized transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ54. Transcriptional activation by the NorR bEBP is controlled by a regulatory GAF domain that represses the ATPase activity of the central AAA+ domain in the absence of nitric oxide. Here, we investigate the mechanism of interdomain repression in NorR by characterizing substitutions in the AAA+ domain that bypass repression by the regulatory domain. Most of these substitutions are located in the vicinity of the surface-exposed loops that engage σ54 during the ATP hydrolysis cycle or in the highly conserved GAFTGA motif that directly contacts σ54. Biochemical studies suggest that the bypass mutations in the GAFTGA loop do not influence the DNA binding properties of NorR or the assembly of higher order oligomers in the presence of enhancer DNA, and as expected these variants retain the ability to activate open complex formation in vitro. We identify a crucial arginine residue in the GAF domain that is essential for interdomain repression and demonstrate that hydrophobic substitutions at this position suppress the bypass phenotype of the GAFTGA substitutions. These observations suggest a novel mechanism for negative regulation in bEBPs in which the GAF domain targets the σ54-interaction surface to prevent access of the AAA+ domain to the sigma factor.

The RNA Polymerase “Switch Region” Is a Target for Inhibitors

The alpha-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"--the hinge that mediates opening and closing of the RNAP active center cleft--to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related alpha-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.

Template Misalignment in Multisubunit RNA Polymerases and Transcription Fidelity

Recent work showed that the single-subunit T7 RNA polymerase (RNAP) can generate misincorporation errors by a mechanism that involves misalignment of the DNA template strand. Here, we show that the same mechanism can produce errors during transcription by the multisubunit yeast RNAP II and bacterial RNAPs. Fluorescence spectroscopy reveals a reorganization of the template strand during this process, and molecular modeling suggests an open space above the polymerase active site that could accommodate a misaligned base. Substrate competition assays indicate that template misalignment, not misincorporation, is the preferred mechanism for substitution errors by cellular RNAPs. Misalignment could account for data previously taken as evidence for additional NTP binding sites downstream of the active site. Analysis of the effects of different template topologies on misincorporation indicates that the duplex DNA immediately downstream of the active site plays an important role in transcription fidelity.

The σ 70 Subunit of RNA Polymerase Is Contacted by the λQ Antiterminator during Early Elongation

The Q protein of bacteriophage λ is a transcription antiterminator that modifies the elongation properties of E. coli RNA polymerase (RNAP). To do this, DNA-bound λQ must first engage a paused elongation complex. Here we show that this engagement of λQ with RNAP involves an interaction between λQ and σ 70, demonstrating that σ 70 can be a target of regulation during elongation. Furthermore, we provide evidence that this interaction between λQ and σ 70 stabilizes a conformation of RNAP that requires the disengagement of a segment of σ 70 from the core enzyme. Recent structure-based models posit that the transition from the initiation to the elongation phase of transcription involves the staged displacement of σ 70 from the RNAP core. Our findings provide support for this proposal.

Isomerization of a binary sigma-promoter DNA complex by transcription activators.

Multisubunit RNA polymerases are targets of sophisticated signal transduction pathways that link environmental or temporal cues to changes in gene expression. Here we show that the sigma 54 protein (sigma54), responsible for promoter specific binding by bacterial RNA polymerase, undergoes a nucleotide hydrolysis dependent isomerization on DNA. Changes in protein structure are evident. The isomerization has all the known requirements of sigma 54-dependent transcription, including a dependence on enhancer binding activator proteins and occurs independently of the core RNA polymerase. We suggest that activator driven changes in sigma54 conformation trigger the conversion of a transcriptionally silent RNA polymerase conformation to one able to interact productively with template DNA. Our results illustrate the types of changes that must occur for multisubunit complexes to manipulate DNA, and show that transcription activators can remodel key nucleoprotein structures to achieve direct activation of transcription.

The RAP74 subunit of human transcription factor IIF has similar roles in initiation and elongation.

Transcription factor IIF (TFIIF) is a protein allosteric effector for RNA polymerase II during the initiation and elongation phases of the transcription cycle. In initiation, TFIIF induces promoter DNA to wrap almost a full turn around RNA polymerase II in a complex that includes the general transcription factors TATA-binding protein, TFIIB, and TFIIE. During elongation, TFIIF also supports a more active conformation of RNA polymerase II. This conformational model for elongation is supported by three lines of experimental evidence. First, a region within the RNA polymerase II-associating protein 74 (RAP74) subunit of TFIIF (amino acids T154 to M177), a region that is critical for isomerization of the preinitiation complex, is also critical for elongation stimulation. Amino acid substitutions within this region are shown to have very similar effects on initiation and elongation, and mutagenic analysis indicates that L155, W164, N172, I176, and M177 are the most important residues in this region for transcription. Second, TFIIF is shown to have a higher affinity for rapidly elongating RNA polymerase II than for the stalled elongation complex, indicating that RNA polymerase II alternates between active and inactive states during elongation and that TFIIF stimulates elongation by supporting the active conformational state of RNA polymerase II. The deleterious I176A substitution in the critical region of RAP74 decreases the affinity of TFIIF for the active form of the elongation complex. Third, TFIIF is shown by Arrhenius analysis to stimulate elongation by populating an activated state of RNA polymerase II.

Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription.

Pre-steady state kinetics of misincorporation were used to investigate the addition of single nucleotides to nascent RNA by Escherichia coli RNA polymerase during transcription elongation. The results were fit with a branched kinetic mechanism that permits conformational switching, at each template position, between an activated and an unactivated enzyme complex, both of which can bind nucleotide triphosphates (NTPs) from solution. The complex exists most often in the long-lived activated state, and only becomes unactivated when transcription is slowed. This model permits multiple levels of nucleotide discrimination in transcription, since the complex can be "kinetically trapped" in the unactivated state in the absence of the correct NTP or if the 3' terminal residue is incorrectly matched. The transcription cleavage factor GreA (or an activity enhanced by GreA) increased the fidelity of transcription by preferential cleavage of transcripts containing misincorporated residues in the unactivated state of the elongation complex. This cleavage mechanism by GreA may prevent the formation of "dead-end" transcription complexes in vivo.

Effects of ppGpp on transcription by DNA-dependent RNA polymerase from Escherichia coli: circular dichroism, absorption and specific transcription studies

Concrete evidence is presented for conformational changes elicited in RNA polymerase upon binding ppGpp by circular dichroism measurements. In the presence of 100 μM ppGpp, the molar ellipticity of RNA polymerase at 220 nm is reduced by 14% from the initial value of −11 100 deg·cm 2·dmol −1 at 25°C. In vitro transcription on templates containing the β-lactamase promoter and colicin E1 promoter on poly[d(A-T)] is inhibited by ppGpp. None of these templates had GC-rich nucleotide sequence near the transcription initiation site, and yet they were influenced by ppGpp. Comparison of the effect on the synthesis of mRNAs for β-lactamase and colicin E1 and the synthesis of the proteins themselves indicates that the effect of ppGpp is at the level of transcription for the former case and involves coupled transcription-translation for the latter case. Difference absorption, polyacrylamide gel electrophoresis, and nitrocellulose filter-binding studies show that the binding of ppGpp to RNA polymerase does not impair the extent of the interaction between enzyme and DNA. Kinetic studies suggest that ppGpp affects transcription initiation on β-lactamase promoter. On poly[d(A-T)], ppGpp affects the rate of open complex formation and is a mixed inhibitor with respect to the incorporation of nucleotides. Our results are consistent with the idea that ppGpp acts as a regulator by binding at a site different from the active site and changes the RNA polymerase conformation, causing altered transcriptional behavior on different DNA templates.

Conditional rifampicin sensitivity of arif mutant ofEscherichia coli: rifampicin induced changes in transcription specificity

Arif mutantof Escherichia coli that exhibits medium and temperature-dependent sensitivity to rifampicin is described. In the absence of rifampicin, this strain grows in minimal and rich media at 30°C and 42°C. In its presence it is viable in rich medium at both temperatures, but in minimal medium only at 30°C. In minimal-rifampicin medium at the higher temperature, RNA synthesis is decreased. The addition of certain divalent salts (MgSO4, CaCl2, BaCl2) in excess, or chelators (EDTA, EGTA, o-phenanthrolein) greatly increase viability in minimal-rifampicin medium at 42°C. Excess MgSO4 (10 mM) also increases the rate of RNA synthesis in the same medium. A model is proposed wherein therif mutation is suggested to cause a structural change in RNA polymerase that allows the binding of rifampicin and other ligands at 42°C. Rifampicin-binding is suggested to alter the conformation of RNA polymerase, impairing its ability to express genes required for growth in minimal medium. Implicit in this view is the assumption that these genes are structurally different from those expressed in rich medium in respect of certain template features recognized by RNA polymerase.