Surface Of RNA Polymerase Research Articles

Analysis of the PcrA-RNA polymerase complex reveals a helicase interaction motif and a role for PcrA/UvrD helicase in the suppression of R-loops.

The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis for this interaction and its functional significance have remained unclear. In this work, we used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation complex in a manner dependent on protein:protein interaction with the conserved PcrA C-terminal Tudor domain. The helicase binds predominantly to two positions on the surface of RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert within the β subunit which we identify as a helicase interaction motif present in many other PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop suppression factor that helps to minimize conflicts between transcription and other processes on DNA including replication.

The mechanism of transcription antitermination by RfaH

RfaH is a bacterial antitermination factor whose function depends on a 12‐nt ops element located in the leader regions of several operons; the ops element both induces RNA polymerase (RNAP) pausing and serves as a recognition site for RfaH. During recruitment, RfaH establishes transient contacts with the ops bases in the non‐template DNA strand exposed on the RNAP surface. After recruitment, RfaH remains bound to RNAP throughout transcription of the entire operon and switches RNAP into a processive state using four mechanisms. First, RfaH competes with σ and prevents σ‐dependent arrest during elongation. Second, RfaH competes with NusG and thus indirectly reduces Rho‐dependent termination. Third, RfaH induces forward translocation of RNAP, thereby inhibiting isomerization into an off‐pathway state that occurs from a pre‐translocated conformation. Fourth, RfaH establishes simultaneous contacts with the β’ clamp and the β lobe, the two elements that maintain the RNAP grip on the nucleic acid chains, thereby preventing the clamp opening that is thought to occur during pausing. While the first three mechanisms are specific to the RfaH‐like proteins, the last mechanism is likely broadly conserved. We propose that structurally diverse antiterminators from bacteria to humans solidify the transcription elongation complex to inhibit conformational transitions that underlie pausing and termination.

Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase

The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding increases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp(220), Asp(318), and Asp(319) for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzyme-bound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme.

DNA Bendability—;A Novel Feature in E. coli Promoter Recognition

The distribution of deformable base-pair steps in the structure of bacterial promoters is analyzed with respect to their possible structural and functional role. A regular positioning of TA and TG stacks is detected with the best fit period 5.6 bp. This value is interpreted as a half of the sequence period 11.2 bp, somewhat higher than the structural helical repeat of B-DNA (10.55 bp). The difference, +0.65 bp, suggests a sequence-dependent helical writhe of the promoter DNA—;a right-handed superhelix. Apparently, to favour rotational setting of DNA on the surface of RNA polymerase the flexible steps deformable largely towards the grooves, follow the half-period spacing. Such rotational setting is consistent with the DNase I footprinting data. Periodical distribution of deformable base-pair stacks shows negative correlation with the presence of -35 canonical hexamer, suggesting the functional significance of this novel element for promoter recognition. The RNA polymerase—;DNA recognition is discussed as interaction of distributional type that involves many elements of different nature which are in partially compensatory relations.

Structural Analysis of the Bacillus subtilisδ Factor: A protein Polyanion which Displaces RNA from RNA Polymerase

RNA polymerase from Bacillus subtilisis a complex mixture comprising a common core (ββ′α 2), the 20.4 kDa delta (δ) protein, and one of several sigma (σ) specificity factors. The δ protein, together with several truncated variants, has been overproduced and purified from Escherichia coli. It is highly acidic (p I=3.6) and contains two distinct regions, a 13 kDa amino-terminal domain with fairly uniform charge distribution and a glutamate and aspartate residue-rich carboxyl-terminal region. The purified amino-terminal domain (δN) contains 32% alpha-helix and 16% beta-sheet, as judged by circular dichroism analysis. In contrast, an 85 kDa tryptic fragment containing the carboxyl-terminal region (δC) is largely unstructured and highly charged (net charge of −47). RNA polymerase purified from a B. subtilismutant with an insertion in the δ gene ( rpoE::cat) contain a truncated δ protein, indicating that the amino-terminal domain is stable in vivoand contains a core-binding function. Addition of δ, but not σ Aof δN, displaces RNA bound to RNA polymerase in a binary complex. The ability of δ to displace RNA efficiently requires the activities of both the amino-terminal core-binding domain and the polyanionic carboxyl-terminal region. Although δC can also displace nucleic acid from RNA polymerase, this activity requires the addition of a large molar excess of protein and is relatively non specific in that both DNA and RNA are displaced. This suggests that the function of the amino-terminal domain is to bind and orient the carboxyl-terminal region on the surface of RNA polymerase.

The nut site of bacteriophage lambda is made of RNA and is bound by transcription antitermination factors on the surface of RNA polymerase.

The boxA and boxB components of the lambda nut site are important for transcriptional antitermination by the phage N protein. We show here that boxA and boxB RNA in N-modified transcription complexes are inaccessible to ribonucleases and have altered sensitivity to dimethylsulfate. N and NusA suffice to weakly protect boxB, independently of boxA and other factors. However, efficient protection of the entire nut site from ribonucleases requires boxA and boxB, N, NusA, NusB, S10, and NusG. Mutations in RNA polymerase, which inhibit antitermination by N in vivo, disallow protection of the nut site during transcription in vitro; therefore, the surface of RNA polymerase must coordinate the formation of complexes containing the antitermination factors and nut site RNA.

Assembly of transcription elongation complexes containing the N protein of phage lambda and the Escherichia coli elongation factors NusA, NusB, NusG, and S10.

The transcription antitermination protein, N, of bacteriophage lambda; the Escherichia coli elongation factors NusA, NusB, ribosomal protein S10, and NusG; and a DNA template containing a lambda nut (N-ututilization) site are necessary and sufficient for the highly cooperative formation in vitro of stable transcription complexes containing all five elongation factors. Mutations in the nut site, NusA, or the beta-subunit of RNA polymerase (RNAP) that impair antitermination in vivo also abolish the assembly of a stable complex containing the antitermination factors in vitro. The effects of RNAP mutations on assembly imply that the antitermination factors assemble on the surface of RNAP. We have shown previously that NusA binds directly to transcribing RNAP (Ka approximately 10(7) M-1); Ka = association constant and we show here that S10 also binds directly and specifically to RNAP with an apparent Ka of 10(6) M-1. These observations led to a model for the ordered assembly of the N-modified transcription complex.