Yuri A. Nedialkov

Transient Reversal of RNA Polymerase II Active Site Closing Controls Fidelity of Transcription Elongation

To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro. The mutation does not appear to significantly affect translocation state equilibrium of Pol II in a stalled elongation complex. Primarily, it promotes NTP sequestration in the polymerase active center. Furthermore, pre-steady-state analyses revealed that the E1103G mutation shifted the equilibrium between the closed and the open active center conformations toward the closed form. Thus, open conformation of the active center emerges as an intermediate essential for preincorporation fidelity control. Similar mechanisms may control fidelity of DNA-dependent DNA polymerases and RNA-dependent RNA polymerases.

NTP-driven Translocation by Human RNA Polymerase II

We report a “running start, two-bond” protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis δ antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure.

Rpb9 Subunit Controls Transcription Fidelity by Delaying NTP Sequestration in RNA Polymerase II

Rpb9 is a small non-essential subunit of yeast RNA polymerase II located on the surface on the enzyme. Deletion of the RPB9 gene shows synthetic lethality with the low fidelity rpb1-E1103G mutation localized in the trigger loop, a mobile element of the catalytic Rpb1 subunit, which has been shown to control transcription fidelity. Similar to the rpb1-E1103G mutation, the RPB9 deletion substantially enhances NTP misincorporation and increases the rate of mismatch extension with the next cognate NTP in vitro. Using pre-steady state kinetic analysis, we show that RPB9 deletion promotes sequestration of NTPs in the polymerase active center just prior to the phosphodiester bond formation. We propose a model in which the Rpb9 subunit controls transcription fidelity by delaying the closure of the trigger loop on the incoming NTP via interaction between the C-terminal domain of Rpb9 and the trigger loop. Our findings reveal a mechanism for regulation of transcription fidelity by protein factors located at a large distance from the active center of RNA polymerase II.

RNA polymerase stalls in a post-translocated register and can hyper-translocate.

Exonuclease (Exo) III was used to probe translocation states of RNA polymerase (RNAP) ternary elongation complexes (TECs). Escherichia coli RNAP stalls primarily in a post-translocation register that makes relatively slow excursions to a hyper-translocated state or to a pre-translocated state. Tagetitoxin (TGT) strongly inhibits hyper-translocation and inhibits backtracking, so, as indicated by Exo III mapping, TGT appears to stabilize both the pre- and probably a partially post-translocation state of RNAP. Because the pre-translocated to post-translocated transition is slow at many template positions, these studies appear inconsistent with a model in which RNAP makes frequent and rapid (i.e., millisecond phase) oscillations between pre- and post-translocation states. Nine nucleotides (9-nt) and 10-nt TECs, and TECs with longer nascent RNAs, have distinct translocation properties consistent with a 9–10 nt RNA/DNA hybrid. RNAP mutant proteins in the bridge helix and trigger loop are identified that inhibit or stimulate forward and backward translocation.

Assay of Transient State Kinetics of RNA Polymerase II Elongation

Publisher Summary This chapter outlines an approach for transient state kinetic analysis of elongation by human RNA polymerase II. For human RNA polymerase II, such a complete analysis might not be possible however highly reliable rate data that are informative for inferring essential aspects of mechanism can be obtained. In the presence of elongation factors, specific steps that are targets of regulation can be identified. Currently, this approach is most applicable to factors demonstrated to interact directly with the elongation complex to stimulate, repress, or edit RNA synthesis. Such factors include transcription factor IIF (TFIIF), the TFIIF-interacting component of the CTD phosphatase (FCP1), DRB sensitivity-inducing factor (DSIF), negative elongation factor (NELF), ELL (a leukemia chromosome translocation partner), SIII/elongin, Cockayne syndrome type B protein (CSB), hepatitis delta antigen (HDAg), and SII/TFIIS. So far, the method is validated by analyzing stimulation of RNA polymerase II by TFIIF and HDAg and inhibition by the mushroom toxin α-amanitin. Transient state kinetic analysis is further shown to have utility for analysis of two regulatory human elongation factors, one of which, HDAg, is associated with severe manifestations of hepatitis B infection.

Translocation and fidelity of Escherichia coli RNA polymerase.

Exonuclease (exo) III was used as a probe of the Escherichia coli RNA polymerase (RNAP) ternary elongation complex (TEC) downstream border. In the absence of NTPs, RNAP appears to stall primarily in a post-translocated state and to return slowly to a pre-translocated state. Exo III mapping, therefore, appears inconsistent with an unrestrained thermal ratchet model for translocation, in which RNAP freely and rapidly oscillates between pre- and post-translocated positions. The forward translocation state is made more stable by lowering the pH and/or by elevating the salt concentration, indicating a probable role of protonated histidine(s) in regulating accurate NTP loading and translocation. Because the post-translocated TEC can be strongly stabilized by NTP addition, NTP analogs were ranked for their ability to preserve the post-translocation state, giving insight into RNAP fidelity. Effects of NTPs (and analogs) and analysis of chemically modified RNA 3′ ends demonstrate that patterns of exo III mapping arise from intrinsic and subtle alterations at the RNAP active site, far from the site of exo III action.

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

Significance The nontemplate DNA strand in the transcription bubble interacts with RNA polymerase and accessory factors to control initiation, elongation, transcription-coupled repair, and translation. During initiation, σ subunit interactions with the nontemplate DNA regulate promoter complex formation and lifetime, abortive synthesis, and start site selection. Here, we show that the β subunit gate loop contacts with an adjacent segment of the nontemplate discriminator region play a similar role during initiation. The deletion of the gate loop alters the structure and properties of promoter complexes and has pleiotropic effects on RNA chain elongation and termination. We propose that, acting in concert with accessory factors, the gate loop mediates the clamp closure and guides the nontemplate strand in initiation and elongation complexes. Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β′ clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

Purification and protein interaction assays of the VP16C transcription activation domain.

Publisher Summary This chapter reviews the many studies of transcriptional activation that have employed the chimeric protein Gal4-VP16, 31 in which the DNA-binding domain of the yeast transcription factor Gal4 fused to the TAD of VP16 is shown. Gal4-VP16 and deletion or substitution mutants thereof have often been used in genetic and biochemical screens for target proteins and activities. The chapter describes a method for the purification of Gal4-VP16C that results in improved yield and quality compared with methods described previously for Gal4-VP1632 or Gal4-VP16C.7, 16 A procedures for normalizing concentrations of Gal4- VP16C protein samples using a sandwich ELISA assay is detailed. It describes the use of surface plasmon resonance (SPR) assays for quantitatively assessing the interactions of Gal4-VP16C, and substitutions mutants thereof with target proteins. Finally, the chapter provides a method for purifying the VP16C polypeptide, separate from any DNA-binding or purification tags, in yields and purity that may be appropriate for a range of structural analyses.

The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand.

RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

Locking the nontemplate DNA to control transcription: RfaH remodels DNA in the transcription elongation complex

Universally conserved NusG/Spt5 factors reduce RNA polymerase pausing and arrest. In a widely accepted model, these proteins bridge the RNA polymerase clamp and lobe domains across the DNA channel, inhibiting the clamp opening to promote pause‐free RNA synthesis. However, recent structures of paused transcription elongation complexes show that the clamp does not open and suggest alternative mechanisms of antipausing. Among these mechanisms, direct contacts of NusG/Spt5 proteins with the nontemplate DNA in the transcription bubble have been proposed to prevent unproductive DNA conformations and thus inhibit arrest. We used Escherichia coli RfaH, whose interactions with DNA are best characterized, to test this idea. We report that RfaH stabilizes the upstream edge of the transcription bubble, favoring forward translocation, and protects the upstream duplex DNA from exonuclease cleavage. Modeling suggests that RfaH loops the nontemplate DNA around its surface and restricts the upstream DNA duplex mobility. Strikingly, we show that RfaH‐induced DNA protection and antipausing activity can be mimicked by shortening the nontemplate strand in elongation complexes assembled on synthetic scaffolds. We propose that remodeling of the nontemplate DNA controls recruitment of regulatory factors and R‐loop formation during transcription elongation across all life.