Cellular RNA polymerases (RNAPs) are complex molecular machines that combine catalysis with concerted conformational changes in the active center. Previous work showed that kinking of a hinge region near the C-terminus of the Bridge Helix (BH-HC) plays a critical role in controlling the catalytic rate.
Here, new evidence for the existence of an additional hinge region in the amino-terminal portion of the Bridge Helix domain (BH-HN) is presented. The nanomechanical properties of BH-HN emerge as a direct consequence of the highly conserved primary amino acid sequence. Mutations that are predicted to influence its flexibility cause corresponding changes in the rate of the nucleotide addition cycle (NAC). BH-HN displays functional properties that are distinct from BH-HC, suggesting that conformational changes in the Bridge Helix control the NAC via two independent mechanisms.
The properties of two distinct molecular hinges in the Bridge Helix of RNAP determine the functional contribution of this domain to key stages of the NAC by coordinating conformational changes in surrounding domains.
During elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile “trigger loop” of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the “bridge helix” that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing.
All atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as “switch” residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop.
Switching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation.
The bridge α-helix in the β′ subunit of RNA polymerase (RNAP) borders the active site and may have roles in catalysis and translocation. In Escherichia coli RNAP, a bulky hydrophobic segment near the N-terminal end of the bridge helix is identified (β′ 772-YFI-774; the YFI motif). YFI is located at a distance from the active center and adjacent to a glycine hinge (β′ 778-GARKG-782) involved in dynamic bending of the bridge helix. Remarkably, amino acid substitutions in YFI significantly alter intrinsic termination, pausing, fidelity and translocation of RNAP. F773V RNAP largely ignores the λ tR2 terminator at 200 µM NTPs and is strongly reduced in λ tR2 recognition at 1 µM NTPs. F773V alters RNAP pausing and backtracking and favors misincorporation. By contrast, the adjacent Y772A substitution increases fidelity and exhibits other transcriptional defects generally opposite to those of F773V. All atom molecular dynamics simulation revealed two separate functional connections emanating from YFI explaining the distinct effects of substitutions: Y772 communicates with the active site through the link domain in the β subunit, whereas F773 communicates through the fork domain in the β subunit. I774 interacts with the F-loop, which also contacts the glycine hinge of the bridge helix. These results identified negative and positive circuits coupled at YFI and employed for regulation of catalysis, elongation, termination and translocation.
RNA polymerase; Bridge helix; Termination; Pausing; Transcriptional fidelity; Translocation
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.
The need for new antibiotics is becoming increasingly critical, as more and more bacteria become resistant to existing drugs. To develop new treatments, researchers need to understand how antibiotics work. One way antibiotics can kill bacteria is by targeting an enzyme called bacterial RNA polymerase. This enzyme builds chains of RNA that bacteria need to survive.
Sal is an antibiotic produced by a marine bacterium found on the surface of a species of jellyfish. Degen, Feng et al. show that Sal kills bacteria by inhibiting bacterial RNA polymerase and explain how Sal inhibits RNA polymerase. Sal binds to a rod-like structural element within RNA polymerase known as the ‘bridge helix’. The bridge helix has been proposed by others to contain two ‘hinges’ that open and close—allowing the bridge helix to bend and unbend—at specific steps in the cycle through which RNA polymerase builds an RNA chain. Degen, Feng et al. show that Sal binds directly to one of the two hinges and show that Sal binds to the hinge in the unbent state. Therefore, Degen, Feng et al. propose that Sal inhibits the enzyme by preventing the hinge from bending.
The binding site on RNA polymerase for Sal is different from, and does not overlap, the binding sites of current antibacterial drugs. As a result, Sal is able to kill bacteria that are resistant to current antibacterial drugs. When Degen, Feng et al. administered Sal in combination with a current antibacterial drug that targets RNA polymerase, bacteria did not detectably develop resistance to either Sal or the current antibacterial drug.
The structure of the complex between Sal and RNA polymerase suggests several ways that Sal could be modified to improve its ability to interact with RNA polymerase, thereby potentially increasing Sal's antibacterial activity. Future research could develop a range of new drugs based on Sal that could kill bacteria more effectively.
RNA polymerase; transcription; inhibitor; antibiotic; bridge helix; bridge-helix cap; E. coli
Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle. To further evaluate the role of the BH, we conducted systematic alanine scanning mutagenesis of the Escherichia coli RNAP BH to determine its contributions to activities required for transcription. Combining our data with an atomic model of E. coli RNAP, we suggest that alterations in the interactions between the BH and (i) the trigger loop, (ii) fork loop 2, and (iii) switch 2 can help explain the observed changes in RNAP functionality associated with some of the BH variants. Additionally, we show that extensive defects in E. coli RNAP functionality depend upon a single previously not studied lysine residue (Lys-781) that is strictly conserved in all bacteria. It appears that direct interactions made by the BH with other conserved features of RNAP are lost in some of the E. coli alanine substitution variants, which we infer results in conformational changes in RNAP that modify RNAP functionality.
Gene Expression; RNA Polymerase; RNA Synthesis; Transcription; Transcription Initiation Factors; RNA Cleavage
Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β’ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.
The archaeal RNA polymerase (RNAP) shares structural similarities with eukaryotic RNAP II but requires a reduced subset of general transcription factors for promoter-dependent initiation. To deepen our knowledge of cellular transcription, we have determined the structure of the 13-subunit DNA-directed RNAP from Sulfolobus shibatae at 3.35 Å resolution. The structure contains the full complement of subunits, including RpoG/Rpb8 and the equivalent of the clamp-head and jaw domains of the eukaryotic Rpb1. Furthermore, we have identified subunit Rpo13, an RNAP component in the order Sulfolobales, which contains a helix-turn-helix motif that interacts with the RpoH/Rpb5 and RpoA′/Rpb1 subunits. Its location and topology suggest a role in the formation of the transcription bubble.
Transcription, the process of converting DNA into RNA (which in turn is translated into proteins by ribosomes) is carried out by the multisubunit RNA polymerase (RNAP) enzyme. Transcription is fundamental to all organisms across the three kingdoms of life—Eukarya, Bacteria, and Archaea—and can be divided into three major steps: initiation, transcription/elongation, and termination. Eukaryotes have three different nuclear RNAPs, whereas Archaea and Bacteria have one. Archaeal transcription is similar to that of eukaryotes, but initiation requires only two accessory proteins bound to DNA: transcription factor B (TFB) and TATA-box binding protein (TBP). It is believed that studies of the archaeal enzyme may shed light on the more complex eukaryotic RNAP. Our complete structure of the archaeal RNAP from Sulfolobus shibatae has fully elucidated its architecture, confirming its close evolutionary relationship with the eukaryotic RNAP II and at the same time revealed a new subunit, Rpo13, with no ortholog in the eukaryotic enzyme. The location and topology of Rpo13 allow us to suggest a mechanism by which Archaea bypass the additional eukaryotic cofactors required for transcription initiation.
The complete archaeal RNA polymerase structure is strikingly similar to the equivalent eukaryotic enzyme. Rpo13, a newly identified archaeal-specific subunit, may explain the minimal set of cofactors required for archaeal transcription initiation.
Transcriptional pausing by multi-subunit RNA Polymerases (RNAPs) is a key mechanism for regulating gene expression in both prokaryotes and eukaryotes, and is a prerequisite for transcription termination. Pausing and termination states are thought to arise through a common, elemental pause state that is inhibitory for nucleotide addition. We report three crystal structures of Thermus RNAP elemental paused elongation complexes (ePECs). The structures reveal the same relaxed, open-clamp RNAP conformation in the ePEC that may arise by failure to reestablish DNA contacts during translocation. A kinked bridge-helix sterically blocks the RNAP active site, explaining how this conformation inhibits RNAP catalytic activity. Our results provide a framework for understanding how RNA hairpin formation stabilizes the paused state and how the ePEC intermediate facilitates termination.
The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (RNAP) II counterpart, comprising a RNAPII-like enzyme as well as two general transcription factors, the TATA-binding protein (TBP) and the eukaryotic TFIIB ortholog TFB1,2. It has been widely understood that precise comparisons among cellular RNAP crystal structures could reveal structural elements common to all enzymes and that these insights would be useful to analyze components of each enzyme that enable it to perform domain-specific gene expression. However, the structure of archaeal RNAP has been limited to individual subunits3,4. Here, we report the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 Å resolution, completing the suite of multi-subunit RNAP structures from all three domains of life. We also report the high resolution (at 1.76 Å) crystal structure of the D/L subcomplex of archaeal RNAP and provide the first experimental evidence of any RNAP possessing an iron-sulfur (Fe-S) cluster, which may play a structural role in a key subunit of RNAP assembly. The striking structural similarity between archaeal RNAP and eukaryotic RNAPII highlights the simpler archaeal RNAP as an ideal model system for dissecting the molecular basis of eukaryotic transcription.
High-resolution crystallographic structures of multisubunit RNA polymerases (RNAPs) have increased our understanding of transcriptional mechanisms. Based on a thorough review of the literature, we have compiled the mutations affecting the function of multisubunit RNA polymerases, many of which having been generated and studied prior to the publication of the first high-resolution structure, and highlighted the positions of the altered amino acids in the structures of both the prokaryotic and eukaryotic enzymes. The observations support many previous hypotheses on the transcriptional process, including the implication of the bridge helix and the trigger loop in the processivity of RNAP, the importance of contacts between the RNAP jaw-lobe module and the downstream DNA in the establishment of a transcription bubble and selection of the transcription start site, the destabilizing effects of ppGpp on the open promoter complex, and the link between RNAP processivity and termination. This study also revealed novel, remarkable features of the RNA polymerase catalytic mechanisms that will require additional investigation, including the putative roles of fork loop 2 in the establishment of a transcription bubble, the trigger loop in start site selection, and the uncharacterized funnel domain in RNAP processivity.
Cellular RNA polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. Two domains in particular, the bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. The functional relevance of these structural changes has been difficult to assess from the relatively small number of static crystal structures currently available.
Using a novel robotic approach we characterized the functional properties of 367 site-directed mutants of the Methanocaldococcus jannaschii RNA polymerase A' subunit, revealing a wide spectrum of in vitro phenotypes. We show that a surprisingly large number of single amino acid substitutions in the bridge helix, including a kink-inducing proline substitution, increase the specific activity of RNA polymerase. Other 'superactivating' substitutions are located in the adjacent base helices of the trigger loop.
The results support the hypothesis that the nucleotide addition cycle involves a kinked bridge helix conformation. The active center of RNA polymerase seems to be constrained by a network of functional interactions between the bridge helix and trigger loop that controls fundamental parameters of RNA synthesis.
Bacterial RNA polymerase (RNAP) is a validated target for antibacterial drugs. CBR703 series antimicrobials allosterically inhibit transcription by binding to a conserved α helix (β′ bridge helix, BH) that interconnects the two largest RNAP subunits. Here we show that disruption of the BH-β subunit contacts by amino-acid substitutions invariably results in accelerated catalysis, slowed-down forward translocation and insensitivity to regulatory pauses. CBR703 partially reverses these effects in CBR-resistant RNAPs while inhibiting catalysis and promoting pausing in CBR-sensitive RNAPs. The differential response of variant RNAPs to CBR703 suggests that the inhibitor binds in a cavity walled by the BH, the β′ F-loop and the β fork loop. Collectively, our data are consistent with a model in which the β subunit fine tunes RNAP elongation activities by altering the BH conformation, whereas CBRs deregulate transcription by increasing coupling between the BH and the β subunit.
Bacterial RNA polymerase (RNAP) is crucial for cellular gene expression and a validated target for antimicrobial drugs. Here, Malinen et al. explore the effects of the CBR class of RNAP inhibitors on the E. coli RNAP transcription cycle and provide detailed mechanistic insight into their antibacterial action.
The in-depth structure/function analysis of large protein complexes, such as RNA polymerases (RNAPs), requires an experimental platform capable of assembling variants of such enzymes in large numbers in a reproducible manner under defined in vitro conditions. Here we describe a streamlined and integrated protocol for assembling recombinant archaeal RNAPs in a high-throughput 96-well format. All aspects of the procedure including construction of redesigned expression plasmids, development of automated protein extraction/in vitro assembly methods and activity assays were specifically adapted for implementation on robotic platforms. The optimized strategy allows the parallel assembly and activity assay of 96 recombinant RNAPs (including wild-type and mutant variants) with little or no human intervention within 24 h. We demonstrate the high-throughput potential of this system by evaluating the side-chain requirements of a single amino acid position of the RNAP Bridge Helix using saturation mutagenesis.
We define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic-acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to, but not overlapping, the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist, and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents.
The trigger loop (TL) in the RNA polymerase (RNAP) active center plays key roles in the reactions of nucleotide addition and RNA cleavage catalyzed by RNAP. The adjacent F loop (FL) was proposed to contribute to RNAP catalysis by modulating structural changes in the TL. Here, we investigate the interplay between these two elements during transcription by bacterial RNAP. Thermodynamic analysis of catalysis by RNAP variants with mutations in the TL and FL suggests that the TL is the key element required for temperature activation in RNAP catalysis, and that the FL promotes TL transitions during nucleotide addition. We reveal characteristic differences in the catalytic parameters between thermophilic Thermus aquaticus and mesophilic Deinococcus radiodurans RNAPs and identify the FL as an adaptable element responsible for the observed differеnces. Mutations in the FL also significantly affect the rate of intrinsic RNA cleavage in a TL-dependent manner. In contrast, much weaker effects of the FL and TL mutations on GreA-assisted RNA cleavage suggest that the FL-dependent TL transitions are not required for this reaction. Thus, functional interplay between the FL and TL is essential for various catalytic activities of RNAP and plays an adaptive role in catalysis by thermophilic and mesophilic enzymes.
Architecture of the RNA polymerase–Spt4/5 complex and basis of universal transcription processivity
Spt5 and NusG play a conserved role in stimulating RNA polymerase II transcription elongation and processivity. Here, the crystal structure of Spt4/5 bound to the RNA polymerase clamp domain reveals that the factor binds above DNA and RNA in the active centre cleft preventing premature dissociation of the polymerase.
Related RNA polymerases (RNAPs) carry out cellular gene transcription in all three kingdoms of life. The universal conservation of the transcription machinery extends to a single RNAP-associated factor, Spt5 (or NusG in bacteria), which renders RNAP processive and may have arisen early to permit evolution of long genes. Spt5 associates with Spt4 to form the Spt4/5 heterodimer. Here, we present the crystal structure of archaeal Spt4/5 bound to the RNAP clamp domain, which forms one side of the RNAP active centre cleft. The structure revealed a conserved Spt5–RNAP interface and enabled modelling of complexes of Spt4/5 counterparts with RNAPs from all kingdoms of life, and of the complete yeast RNAP II elongation complex with bound Spt4/5. The N-terminal NGN domain of Spt5/NusG closes the RNAP active centre cleft to lock nucleic acids and render the elongation complex stable and processive. The C-terminal KOW1 domain is mobile, but its location is restricted to a region between the RNAP clamp and wall above the RNA exit tunnel, where it may interact with RNA and/or other factors.
gene regulation; gene transcription; multiprotein complex structure; RNA polymerase elongation; transcription elongation factor
RNA polymerase from S. shibatae in complex with DNA was crystallized using a nanoseeding method. Native RNAP crystals underwent an ad hoc procedure for DNA binding; one of these crystals diffracted to 4.3 Å resolution.
Transcription is a fundamental process across the three domains of life and is carried out by multi-subunit enzymatic DNA-directed RNA polymerases (RNAPs). The interaction of RNAP with nucleic acids is tightly controlled for precise and processive RNA synthesis. Whilst a wealth of structural information has been gathered on the eukaryotic Pol II in complex with DNA/RNA, no information exists on its ancestral counterpart archaeal RNAP. Thus, in order to extend knowledge of the archaeal transcriptional apparatus, crystallization of Sulfolobus shibatae RNAP (molecular mass of ∼400 kDa) with DNA fragments was pursued. To achieve this goal, crystal growth was first optimized using a nanoseeding technique. An ad hoc soaking protocol was then put into place, which consisted of gently exchanging the high-salt buffer used for apo-RNAP crystal growth into a low-salt buffer necessary for DNA binding to RNAP. Of the various crystals screened, one diffracted to 4.3 Å resolution and structural analysis showed the presence of bound DNA [Wojtas et al. (2012 ▶). Nucleic Acids Res.
archaeal transcription; RNA polymerases; DNA complexes
Three conserved aspartate residues in the largest subunit of multisubunit RNA polymerases (RNAPs) coordinate two Mg2+ ions involved in the catalysis of phosphodiester bond synthesis. A structural model based on the stereochemistry of nucleotidyl transfer reaction as well as recent crystallographic data predict that these Mg2+ ions should also be involved in the reverse reaction of pyrophosphorolysis as well as in the endo- and exonucleolytic cleavage of the nascent RNA. Here, we check these predictions by constructing point substitutions of each of the three Asp residues in the β′ subunit of Escherichia coli RNAP and testing the mutant enzymes' functions. Using artificially assembled elongation complexes, we demonstrate that substitutions of any of the three aspartates dramatically reduce all known RNAP catalytic activities, supporting the model's predictions that same amino acids participate in all RNAP catalytic reactions. We demonstrate that though substitutions in the DFDGD motif decrease Mg2+ binding to free RNAP below detection limits, the apparent affinity to Mg2+ in transcription complexes formed by the mutant and wild-type RNAPs is similar, suggesting that NTP substrates and/or nucleic acids actively contribute to the retention of active center Mg2+.
Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA–RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5—the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.
The nucleotidyl transfer reaction leading to formation of the first phosphodiester bond has been followed in real-time by Raman microscopy, as it proceeds in single crystals of the N4 phage virion RNA polymerase (RNAP). The reaction is initiated by soaking NTP substrates and divalent cations into the RNAP and promoter DNA complex crystal, where the phosphodiester bond formation is completed in about 40 minutes. This slow reaction allowed us to monitor the changes of RNAP and DNA conformations as well as bindings of substrate and metal through Raman spectra taken every 5 minutes. Recently published snapshot X-ray crystal structures along the same reaction pathway assisted the spectroscopic assignments of changes in the enzyme and DNA, while isotopically labeled NTP substrates allowed differentiation of the Raman spectra of bases in substrates and DNA. We observed that substrates are bound at 2-7 minutes after commencing soaking, the O-helix completes its conformational change, and that binding of both divalent metals required for catalysis in the active site changes the conformation of the ribose triphosphate at position +1. These are followed by a slower decrease of NTP triphosphate groups due to phosphodiester bond formation that reaches completion at about 15 minutes, and even slower complete release of the divalent metals at about 40 minutes. We have also shown that the O-helix movement can be driven by substrate binding only. The kinetics of the in crystallo nucleotidyl transfer reaction revealed in this study suggest that soaking the substrate and metal into the RNAP-DNA complex crystal for a few minutes generates novel and uncharacterized intermediates for future X-ray and spectroscopic analysis.
Tagetitoxin (Tgt) inhibits plastid-encoded, bacterial and some eukaryotic RNA polymerases (RNAPs) by an unknown mechanism. A 2.4Å-resolution structure of the Thermus thermophilus RNAP/Tgt complex revealed that Tgt-binding site within the RNAP secondary channel overlaps with that of the stringent control effector ppGpp, which partially protects RNAP from Tgt inhibition. Tgt binding is mediated exclusively through polar interactions with the β and β′ residues whose substitutions confer resistance to Tgt in vitro. Importantly, a Tgt phosphate, together with two active site acidic residues, coordinates the third Mg2+ ion distinct from the two catalytic metal ions. We show that Tgt inhibits all RNAP catalytic reactions and propose a mechanism in which the Tgt-bound Mg2+ ion plays a key role in stabilization of an inactive transcription intermediate. This and other recent studies suggest that Mg-mediated remodeling of the active site could be a common theme in the regulation of catalysis by nucleic acid enzymes.
In this study, we have identified a unique mechanism in which human cytomegalovirus (HCMV) protein pUL79 acts as an elongation factor to direct cellular RNA polymerase II for viral transcription during late times of infection. We and others previously reported that pUL79 and its homologues are required for viral transcript accumulation after viral DNA synthesis. We hypothesized that pUL79 represented a unique mechanism to regulate viral transcription at late times during HCMV infection. To test this hypothesis, we analyzed the proteome associated with pUL79 during virus infection by mass spectrometry. We identified both cellular transcriptional factors, including multiple RNA polymerase II (RNAP II) subunits, and novel viral transactivators, including pUL87 and pUL95, as protein binding partners of pUL79. Co-immunoprecipitation (co-IP) followed by immunoblot analysis confirmed the pUL79-RNAP II interaction, and this interaction was independent of any other viral proteins. Using a recombinant HCMV virus where pUL79 protein is conditionally regulated by a protein destabilization domain ddFKBP, we showed that this interaction did not alter the total levels of RNAP II or its recruitment to viral late promoters. Furthermore, pUL79 did not alter the phosphorylation profiles of the RNAP II C-terminal domain, which was critical for transcriptional regulation. Rather, a nuclear run-on assay indicated that, in the absence of pUL79, RNAP II failed to elongate and stalled on the viral DNA. pUL79-dependent RNAP II elongation was required for transcription from all three kinetic classes of viral genes (i.e. immediate-early, early, and late) at late times during virus infection. In contrast, host gene transcription during HCMV infection was independent of pUL79. In summary, we have identified a novel viral mechanism by which pUL79, and potentially other viral factors, regulates the rate of RNAP II transcription machinery on viral transcription during late stages of HCMV infection.
In this study, we report a novel mechanism used by human cytomegalovirus (HCMV) to regulate the elongation rate of RNA polymerase II (RNAP II) to facilitate viral transcription during late stages of infection. Recently, we and others have identified several viral factors that regulate gene expression during late infection. These factors are functionally conserved among beta- and gamma- herpesviruses, suggesting a unique transcriptional regulation shared by viruses of these two subfamilies. However, the mechanism remains elusive. Here we show that HCMV pUL79, one of these factors, interacts with RNAP II as well as other viral factors involved in late gene expression. We have started to elucidate the nature of the pUL79-RNAP II interaction, finding that pUL79 does not alter the protein levels of RNAP II or its recruitment to viral promoters. However, during late times of infection, pUL79 helps RNAP II efficiently elongate along the viral DNA template to transcribe HCMV genes. Host genes are not regulated by this pUL79-mediated mechanism. Therefore, our study discovers a previously uncharacterized mechanism where RNAP II activity is modulated by viral factor pUL79, and potentially other viral factors as well, for coordinated viral transcription.
The structure of the final initiation complex between E. coli RNA polymerase (RNAP) and the bla promoter from the transposon TN3 has been probed by footprinting experiments and base accessibility to dimethyl sulfate at 37 degrees C. At RNAP/promoter molar ratios "standard" for these experiments (greater than or equal to 10), the contacts on bla extend from -100 to +20, i.e. a length exceeding twice the dimension of the RNAP major axis . Since footprinting at about equimolar amounts of RNAP and bla extends to the usual (-55 to +20) promoter domain, it is very likely that at least two RNAP's participate in the complex observed at tenfold higher RNAP/bla ratios. Under the latter conditions, the extended footprint (-100 to +20) is observed above 30 degrees C, whereas at 15 degrees C, only the -55 to +20 promoter area is contacted. Furthermore, gel retardation experiments show the presence of two complexes of different migration rates. We have reported earlier  that at the "standard" RNAP/bla ratio, transcription initiation from the bla promoter is inhibited. The correlation of this inhibition with the postulated two RNAP/bla complex suggests a regulation of bla gene expression by RNAP availability controlled for instance by growth rate. These results can be correlated with those reported in [14, 15] for the tyrT promoter. Interestingly, both promoter share significant sequence homologies.
Multi-subunit RNA polymerases (RNAPs) in all three domains of life share a common ancestry. The composition of the archaeal RNAP (aRNAP) is not identical between phyla and species, with subunits Rpo8 and Rpo13 found in restricted subsets of archaea. While Rpo8 has an ortholog, Rpb8, in the nuclear eukaryal RNAPs, Rpo13 lacks clear eukaryal orthologs. Here, we report crystal structures of the DNA-bound and free form of the aRNAP from Sulfolobus shibatae. Together with biochemical and biophysical analyses, these data show that Rpo13 C-terminus binds non-specifically to double-stranded DNA. These interactions map on our RNAP–DNA binary complex on the downstream DNA at the far end of the DNA entry channel. Our findings thus support Rpo13 as a RNAP–DNA stabilization factor, a role reminiscent of eukaryotic general transcriptional factors. The data further yield insight into the mechanisms and evolution of RNAP–DNA interaction.
A combination of structural approaches yields a complete atomic model of the highly biochemically characterized Escherichia coli RNA polymerase, enabling fuller exploitation of E. coli as a model for understanding transcription.
The Escherichia coli transcription system is the best characterized from a biochemical and genetic point of view and has served as a model system. Nevertheless, a molecular understanding of the details of E. coli transcription and its regulation, and therefore its full exploitation as a model system, has been hampered by the absence of high-resolution structural information on E. coli RNA polymerase (RNAP). We use a combination of approaches, including high-resolution X-ray crystallography, ab initio structural prediction, homology modeling, and single-particle cryo-electron microscopy, to generate complete atomic models of E. coli core RNAP and an E. coli RNAP ternary elongation complex. The detailed and comprehensive structural descriptions can be used to help interpret previous biochemical and genetic data in a new light and provide a structural framework for designing experiments to understand the function of the E. coli lineage-specific insertions and their role in the E. coli transcription program.
Transcription, or the synthesis of RNA from DNA, is one of the most important processes in the cell. The central enzyme of transcription is the DNA-dependent RNA polymerase (RNAP), a large, macromolecular assembly consisting of at least five subunits. Historically, much of our fundamental information on the process of transcription has come from genetic and biochemical studies of RNAP from the model bacterium Escherichia coli. More recently, major breakthroughs in our understanding of the mechanism of action of RNAP have come from high resolution crystal structures of various bacterial, archaebacterial, and eukaryotic enzymes. However, all of our high-resolution bacterial RNAP structures are of enzymes from the thermophiles Thermus aquaticus or T. thermophilus, organisms with poorly characterized transcription systems. It has thus far proven impossible to obtain a high-resolution structure of E. coli RNAP, which has made it difficult to relate the large collection of genetic and biochemical data on RNAP function directly to the available structural information. Here, we used a combination of approaches—high-resolution X-ray crystallography of E. coli RNAP fragments, ab initio structure prediction, homology modeling, and single-particle cryo-electron microscopy—to generate complete atomic models of E. coli RNAP. Our detailed and comprehensive structural models provide the heretofore missing structural framework for understanding the function of the highly characterized E. coli RNAP.