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Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the −10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation. Inside cells, molecules of double-stranded DNA encode the instructions needed to make proteins. To make a protein, the two strands of DNA that make up a gene are separated and one strand acts as a template to make molecules of messenger ribonucleic acid (or mRNA for short). This process is called transcription. The mRNA is then used as a template to assemble the protein. An enzyme called RNA polymerase carries out transcription and is found in all cells ranging from bacteria to humans and other animals. Bacteria have the simplest form of RNA polymerase and provide an excellent system to study how it controls transcription. It is made up of several proteins that work together to make RNA using DNA as a template. However, it requires the help of another protein called sigma factor to direct it to regions of DNA called promoters, which are just before the start of the gene. When RNA polymerase and the sigma factor interact the resulting group of proteins is known as the RNA polymerase ‘holoenzyme’. Transcription takes place in several stages. To start with, the RNA polymerase holoenzyme locates and binds to promoter DNA. Next, it separates the two strands of DNA and exposes a portion of the template strand. At this point, the DNA and the holoenzyme are said to be in an ‘open promoter complex’ and the section of promoter DNA that is within it is known as a ‘transcription bubble’. However, it is not clear how RNA polymerase holoenzyme interacts with DNA in the open promoter complex. Bae, Feklistov et al. have now used X-ray crystallography to reveal the three-dimensional structure of the open promoter complex with an entire transcription bubble from a bacterium called Thermus aquaticus. The experiments show that there are several important interactions between RNA polymerase holoenzyme and promoter DNA. In particular, the sigma factor inserts into a region of the DNA at the start of the transcription bubble. This rearranges the DNA in a manner that allows the DNA to be exposed and contact the main part of the RNA polymerase. If the holoenyzyme fails to contact the DNA in this way, the holoenzyme does not bind properly to the promoter and transcription does not start. These findings build on previous work to provide a detailed structural framework for understanding how the RNA polymerase holoenzyme and DNA interact to form the open promoter complex. Another study by Bae et al.—which involved some of the same researchers as this study—reveals how another protein called CarD also binds to DNA at the start of the transcription bubble to stabilize the open promoter complex.

A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex 1 – 3 . To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12–14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed 4 – 6 . Here we present cryo-electron microscopy structures of bacterial RNAP–promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2—universal structural features of RNAP—in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life. Cryo-electron microscopy structures of bacterial RNAP–promoter DNA complexes, including structures of partially melted intermediates, suggest a universally conserved common mechanism for promoter DNA opening prior to gene expression.

We report crystal structures of the antibacterial lasso peptides microcin J25 (MccJ25) and capistruin (Cap) bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). Both peptides bind within the RNAP secondary channel, through which NTP substrates enter the RNAP active site, and sterically block trigger-loop folding, which is essential for efficient catalysis by the RNAP. MccJ25 binds deep within the secondary channel in a manner expected to interfere with NTP substrate binding, explaining the partial competitive mechanism of inhibition with respect to NTPs found previously [Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004) Mol Cell 14:739–751]. The Cap binding determinant on RNAP overlaps, but is not identical to, that of MccJ25. Cap binds further from the RNAP active site and does not sterically interfere with NTP binding, and we show that Cap inhibition is partially noncompetitive with respect to NTPs. This work lays the groundwork for structure determination of other lasso peptides that target the bacterial RNAP and provides a structural foundation to guide lasso peptide antimicrobial engineering approaches.

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA–protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3′ segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3′ end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication–transcription complex or RTC) associated with two copies of nsp13 (nsp13 2 –RTC). One copy of nsp13 interacts with the template-RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here we use cryogenic-electron microscopy and molecular dynamics simulations to analyze the nsp13 2 –RTC, revealing four distinct conformational states of the helicases. The results indicate a mechanism for the nsp13 2 –RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site. In their complex, the SARS-CoV-2 nsp13 helicase and RNA polymerase would translocate on RNA in opposite directions. Cryo-EM and MD simulations resolve this conundrum, suggesting an allosteric mechanism to turn the helicase on and off.

Transcription in bacteria is a multi-step process. In the first step, contacts between RNA polymerase and the promoter DNA must be established for transcription initiation to begin, but then these contacts must be broken for the enzyme to transition into the elongation phase. Single-molecule and biochemical observations report that promoter escape is a highly regulated and sometimes rate-limiting step in the transcription cycle; however, the structural mechanisms of promoter escape remain obscure. Promoter escape also serves as the target for the clinically important antibiotic rifampicin, used to treat tuberculosis. Here, we present seven distinct intermediates showing the structural details of M. tuberculosis RNA polymerase initial transcribing complexes and promoter escape, using a de novo cryo-electron microscopy approach. We describe the structural rearrangements that RNA polymerase undergoes to clear the promoter, including those required to release the initiation factor, σ, providing a structural account for decades of biochemical observations. These structures and supporting biochemistry provide a model of promoter escape, a universal step in the transcription cycle, with conformations that may be used to develop Rifampicin alternatives. Promoter escape is a key step in bacterial transcription and a target of the antibiotic rifampicin. Here, the authors use cryo-EM to explore this step, finding seven structural intermediates of M. tuberculosis RNA polymerase during promoter escape.

Bacterial σ factors bind RNA polymerase (E) to form holoenzyme (Eσ), conferring promoter specificity to E and playing a key role in transcription bubble formation. σ N is unique among σ factors in its structure and functional mechanism, requiring activation by specialized AAA+ ATPases. Eσ N forms an inactive promoter complex where the N-terminal σ N region I (σ N -RI) threads through a small DNA bubble. On the opposite side of the DNA, the ATPase engages σ N -RI within the pore of its hexameric ring. Here, we perform kinetics-guided structural analysis of de novo formed Eσ N initiation complexes and engineer a biochemical assay to measure ATPase-mediated σ N -RI translocation during promoter melting. We show that the ATPase exerts mechanical action to translocate about 30 residues of σ N -RI through the DNA bubble, disrupting inhibitory structures of σ N to allow full transcription bubble formation. A local charge switch of σ N -RI from positive to negative may help facilitate disengagement of the otherwise processive ATPase, allowing subsequent σ N disentanglement from the DNA bubble. Bacterial transcription with σ N requires activation by specialized AAA+ ATPases. Here, the authors visualize transient structural intermediates and engineer a biochemical assay to show that these ATPases partially unfold σ N to initiate transcription.

Bacteriophage T7 encodes an essential inhibitor of the Escherichia coli host RNA polymerase (RNAP), the product of gene 2 (Gp2). We determined a series of X-ray crystal structures of E. coli RNAP holoenzyme with or without Gp2. The results define the structure and location of the RNAP σ ⁷⁰ subunit domain 1.1 [Formula] inside the RNAP active site channel, where it must be displaced by the DNA upon formation of the open promoter complex. The structures and associated data, combined with previous results, allow for a complete delineation of the mechanism for Gp2 inhibition of E. coli RNAP. In the primary inhibition mechanism, Gp2 forms a protein–protein interaction with [Formula], preventing the normal egress of [Formula] from the RNAP active site channel. Gp2 thus misappropriates a domain of the RNAP holoenzyme, [Formula], to inhibit the function of the enzyme.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication–transcription complex (RTC) 1 . Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir 2 . Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate 3 , 4 . Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation 5 . The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5′ RNA cap 6 . Cryo-EM is used to visualize the SARS-CoV-2 RTC bound to each of the natural NTPs as well as remdesivir triphosphate (RDV-TP) in states poised for incorporation, explaining the interactions required for NTP recognition and RDV-TP selectivity.

All cellular RNA polymerases (RNAPs), from those of bacteria to those of man, possess a clamp that can open and close, and it has been assumed that the open RNAP separates promoter DNA strands and then closes to establish a tight grip on the DNA template. Here, we resolve successive motions of the initiating bacterial RNAP by studying real-time signatures of fluorescent reporters placed on RNAP and DNA in the presence of ligands locking the clamp in distinct conformations. We report evidence for an unexpected and obligatory step early in the initiation involving a transient clamp closure as a prerequisite for DNA melting. We also present a 2.6-angstrom crystal structure of a late-initiation intermediate harboring a rotationally unconstrained downstream DNA duplex within the open RNAP active site cleft. Our findings explain how RNAP thermal motions control the promoter search and drive DNA melting in the absence of external energy sources.