The mechanism of substrate loading in multisubunit RNA polymerase is crucial for understanding the general principles of transcription yet remains hotly debated. Here we report the 3.0-A resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-5'-[(alpha,beta)-methyleno]-triphosphate (AMPcPP), and with AMPcPP plus the inhibitor streptolydigin. In the EC/AMPcPP structure, the substrate binds to the active ('insertion') site closed through refolding of the trigger loop (TL) into two alpha-helices. In contrast, the EC/AMPcPP/streptolydigin structure reveals an inactive ('preinsertion') substrate configuration stabilized by streptolydigin-induced displacement of the TL. Our structural and biochemical data suggest that refolding of the TL is vital for catalysis and have three main implications. First, despite differences in the details, the two-step preinsertion/insertion mechanism of substrate loading may be universal for all RNA polymerases. Second, freezing of the preinsertion state is an attractive target for the design of novel antibiotics. Last, the TL emerges as a prominent target whose refolding can be modulated by regulatory factors.

The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-A resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.

SUMMARY NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a β-barrel in NusG and an α-hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here we show that the released RfaH-CTD completely refolds from an all-α to an all-β conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated.

Transcript elongation by RNA polymerase is discontinuous and interrupted by pauses that play key regulatory roles. We show here that two different classes of pause signals punctuate elongation. Class I pauses, discovered in enteric bacteria, depend on interaction of a nascent RNA structure with RNA polymerase to displace the 3 OH away from the catalytic center. Class II pauses, which may predominate in eukaryotes, cause RNA polymerase to slide backwards along DNA and RNA and to occlude the active site with nascent RNA. These pauses differ in their responses to antisense oligonucleotides, pyrophosphate, GreA, and general elongation factors NusA and NusG. In contrast, substitutions in RNA polymerase that increase or decrease the rate of RNA synthesis affect both pause classes similarly. We propose that both pause classes, as well as arrest and termination, arise from a common intermediate that itself binds NTP substrate weakly.transcriptional pausing ͉ NusA ͉ NusG G ene expression often is regulated during RNA chain elongation. Regulation depends both on interruptions to transcription caused by pause, arrest, and termination signals encoded in the DNA and RNA and on auxiliary proteins that modify the response of RNA polymerase (RNAP) to these signals (reviewed in refs. 1 and 2). Pausing (a temporary delay in chain elongation) synchronizes transcription and translation in prokaryotes, slows RNAP to allow timely interaction of regulatory factors, and is a precursor to both arrest (complete halting without dissociation; refs. 3 and 4), and dissociation of the transcription elongation complex (TEC) at -dependent and -independent terminators (1).Numerous auxiliary proteins modulate pausing in organisms from bacteria to humans. Two of these, NusA and NusG, are universally conserved among bacteria and archaebacteria (5), are typically essential to cell viability, and, respectively, inhibit or stimulate pausing by bacterial RNAP (6). NusA and NusG also modulate the termination activity of protein (which dissociates paused TECs) and, together with other auxiliary proteins like N or Q, assemble antitermination TECs that resist pausing and termination.Although transcriptional pausing was first described more than two decades ago (7), no consensus pause sequence exists. Rather, different types of signals appear to inhibit alignment of the RNA 3Ј OH with substrate NTP in different ways (8-12). Most if not all of these signals depend on RNAP's ability to slide back and forth along RNA and DNA chains (while preserving an Ϸ17-nt DNA bubble and Ϸ8-bp RNA⅐DNA hybrid; refs. 4, 13, 14, and references therein). These movements may produce five distinct TEC configurations (see Fig. 1 To clarify the mechanism of pausing and to examine how it is modulated by NusA and NusG, we compared the two types of pause signals that have been studied most extensively, which we call class I and class II pauses. At a class I pause, nascent RNA hairpin-RNAP interaction inhibits nucleotide addition by stabilizing the RNA 3Ј OH in a frayed or hypertranslo...

Bacterial transcription is regulated by the alarmone ppGpp, which binds near the catalytic site of RNA polymerase (RNAP) and modulates its activity. We show that the DksA protein is a crucial component of ppGpp-dependent regulation. The 2.0 A resolution structure of Escherichia coli DksA reveals a globular domain and a coiled coil with two highly conserved Asp residues at its tip that is reminiscent of the transcript cleavage factor GreA. This structural similarity suggests that DksA coiled coil protrudes into the RNAP secondary channel to coordinate a ppGpp bound Mg2+ ion with the Asp residues, thereby stabilizing the ppGpp-RNAP complex. Biochemical analysis demonstrates that DksA affects transcript elongation, albeit differently from GreA; augments ppGpp effects on initiation; and binds directly to RNAP, positioning the Asp residues near the active site. Substitution of these residues eliminates the synergy between DksA and ppGpp. Thus, the secondary channel emerges as a common regulatory entrance for transcription factors.

RfaH, a paralog of the general transcription factor NusG, is recruited to elongating RNA polymerase at specific regulatory sites. The X-ray structure of Escherichia coli RfaH reported here reveals two domains. The N-terminal domain displays high similarity to that of NusG. In contrast, the alpha-helical coiled-coil C domain, while retaining sequence similarity, is strikingly different from the beta barrel of NusG. To our knowledge, such an all-beta to all-alpha transition of the entire domain is the most extreme example of protein fold evolution known to date. Both N domains possess a vast hydrophobic cavity that is buried by the C domain in RfaH but is exposed in NusG. We propose that this cavity constitutes the RNA polymerase-binding site, which becomes unmasked in RfaH only upon sequence-specific binding to the nontemplate DNA strand that triggers domain dissociation. Finally, we argue that RfaH binds to the beta' subunit coiled coil, the major target site for the initiation sigma factors.

Transcription of eukaryotic genes by RNA polymerase II (Pol II) is typically accompanied by nucleosome survival and minimal exchange of histones H3/H4. The mechanism of survival and recovery of chromatin structure remains obscure. Here we show how transcription through chromatin by Pol II is uniquely coupled with nucleosome survival. Structural modeling and functional analysis of the intermediates of transcription through a nucleosome was conducted. When Pol II approaches the area of strong DNA-histone interactions, a small intranucleosomal DNA loop (zero-size or Ø-loop) containing transcribing enzyme is formed. During formation of the Ø-loop, the recovery of DNA-histone interactions behind Pol II is tightly coupled with their disruption ahead of the enzyme. This coupling is a distinct feature of the Pol II-type mechanism that allows further transcription through the nucleosome, prevents nucleosome translocation and minimizes displacement of H3/H4 histones from DNA during enzyme passage.

NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ρ-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.