[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The bridge -helix in the ' 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 (' 772-YFI-774; the YFI motif). YFI is located at a distance from the active center and adjacent to a glycine hinge (' 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 tR2 terminator at 200M NTPs and is strongly reduced in tR2 recognition at 1M 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 subunit, whereas F773 communicates through the fork domain in the 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.
No preview · Article · Nov 2012 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jun 2009 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Strategies for assembly and analysis of human, yeast, and bacterial RNA polymerase elongation complexes are described, and methods are shown for millisecond phase kinetic analyses of elongation using rapid chemical quench flow. Human, yeast, and bacterial RNA polymerases function very similarly in NTP-Mg2+ commitment and phosphodiester bond formation. A "running start, two-bond, double-quench" protocol is described and its advantages discussed. These studies provide information about stable NTP-Mg2+ loading, phosphodiester bond synthesis, the processive transition between bonds, and sequence-specific effects on transcription elongation dynamics.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Multi-subunit RNA polymerases bind nucleotide triphosphate (NTP) substrates in the pretranslocated state and carry the dNMP-NTP base pair into the active site for phosphoryl transfer. NTP-driven translocation requires that NTP substrates enter the main-enzyme channel before loading into the active site. Based on this model, a new view of fidelity and efficiency of RNA synthesis is proposed. The model predicts that, during processive elongation, NTP-driven translocation is coupled to a protein conformational change that allows pyrophosphate release: coupling the end of one bond-addition cycle to substrate loading and translocation for the next. We present a detailed model of the RNA polymerase II elongation complex based on 2 low-affinity NTP binding sites located in the main-enzyme channel. This model posits that NTP substrates, elongation factors, and the conserved Rpb2 subunit fork loop 2 cooperate to regulate opening of the downstream transcription bubble.
[Show abstract][Hide abstract] ABSTRACT: The distance between the TATAAAAG box and initiator element of the strong adenovirus major late promoter was systematically altered to determine the optimal spacing for simultaneous recognition of both elements. We find that the TATAAAAG element is strongly dominant over the initiator for specification of the start site. The wild type spacing of 23 base pairs between TATAAAAG and +1A is optimal for promoter strength and selective recognition of the A-start. Initiation is constrained to a window spaced 19-26 base pairs downstream of (-31)-TATAAAAG-(-24), and A-starts are favored over alternate starts only when spaced between 21 and 25 base pairs downstream of TATAAAAG. We report an expanded TATAAAAG and initiator promoter consensus for vertebrates and plants. Plant promoters of this class are (A-T)-rich and have an A-rich (non-template strand) core promoter sequence element downstream of +1A.
Preview · Article · Apr 2005 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: Our laboratory has developed methods for transient state kinetic analysis of human RNA polymerase II elongation. In these studies, multiple conformations of the RNA polymerase II elongation complex were revealed by their distinct elongation potential and differing dependence on nucleoside triphosphate substrate. Among these are conformations that appear to correspond to different translocation states of the DNA template and RNA-DNA hybrid. Using alpha-amanitin as a dynamic probe of the RNA polymerase II mechanism, we show that the most highly poised conformation of the elongation complex, which we interpreted previously as the posttranslocated state, is selectively resistant to inhibition with alpha-amanitin. Because initially resistant elongation complexes form only a single phosphodiester bond before being rendered inactive in the following bond addition cycle, alpha-amanitin inhibits elongation at each translocation step.
[Show abstract][Hide abstract] ABSTRACT: Models of mechanisms of transcriptional activation in eukaryotes frequently invoke direct interactions of transcriptional activation domains with target proteins including general transcription factors or coactivators such as chromatin modifying complexes. The potent transcriptional activation domain (AD) of the VP16 protein of herpes simplex virus has previously been shown to interact with several general transcription factors including the TATA-binding protein (TBP), TBP-associated factor 9 (TAF9), TFIIA, and TFIIB. In surface plasmon resonance assays, a module of the VP16 AD designated VP16C (residues 452-490) bound to TBP with an affinity notably stronger than to TAF9, TFIIA or TFIIB. Moreover, the interaction of VP16C with TBP correlated well with transcriptional activity for a panel of VP16C substitution variants. These results support models in which the interactions of ADs with TBP play an important role in transcriptional activation.
No preview · Article · Jun 2004 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jun 2003 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Feb 2003 · Methods in Enzymology
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Feb 2003 · Methods in Enzymology
[Show abstract][Hide abstract] ABSTRACT: RNA polymerase II-associating protein 74 (RAP74) is the large subunit of transcription factor IIF (TFIIF), which is essential
for accurate initiation and stimulates elongation by RNA polymerase II. Mutations within or adjacent to the α1 helix of the
RAP74 subunit have been shown to decrease both initiation and elongation stimulation activities without strongly affecting
the interactions of RAP74 with the RAP30 subunit or the interaction between TFIIF and RNA polymerase II. In this manuscript,
mutations within the α1 helix are compared with mutations made throughout the neighboring conserved N-terminal domain of RAP74.
Changes within the N-terminal domain include disruptions of specific contacts with the α1 helix, which were revealed in the
recently published x-ray crystal structure (Gaiser, F., Tan, S., and Richmond, T. J. (2000) J. Mol. Biol. 302, 1119–1127). Contacts between the β4-β5 loop and the α1 helix are shown to be largely unimportant for α1 helix function.
Other mutations throughout the N-terminal domain are consistent with the establishment of the dimer interface with the RAP30
subunit. The RAP74-RAP30 interface is important for TFIIF function, but no particular RAP74 amino acids within this region
have been identified that are required for TFIIF activities. The molecular target of the α1 helix remains unknown, but our
studies refocus attention on this important functional motif of TFIIF.
No preview · Article · Jan 2003 · Journal of Biological Chemistry