title: The intercalating beta-hairpin of T7 RNA polymerase plays a role in promoter DNA melting and in stabilizing the melted DNA for efficient RNA synthesis.
abstract: Phage T7 RNA polymerase contains within its single polypeptide all the elements for specific recognition and melting of its promoter DNA. Crystallographic studies indicate that a beta-hairpin (230-245) with an intercalating valine residue plays a role in promoter opening. We mutated V237 to several amino acids, deleted five amino acid residues at the tip of the hairpin, and mutated E242 and D240 at the base of the hairpin to define the roles of the tip and base of the hairpin in DNA strand separation. The affinity of the hairpin mutants for the promoter DNA was not significantly affected. Stopped-flow kinetic studies showed that the bimolecular rate of DNA binding and the observed rate of pre-initiation open complex formation that corresponds to the sum of DNA opening and closing steps were within 20 to 40 % of the wild-type polymerase. Yet, most mutants showed a smaller amount of the pre-initiation open complex at equilibrium, indicating that the individual rates of promoter opening and closing steps were altered in the mutants. The base mutants, E242A and D240A, showed both a lower rate of promoter opening and a higher rate of promoter closing, suggesting their role in stabilization of the open complex. The V237D and the deletion mutant showed mainly a lower rate of promoter opening, suggesting that the tip of the hairpin may nucleate DNA opening. The defect in pre-initiation open complex formation affected downstream steps such as the rate of the first phosphodiester bond formation step, but did not affect significantly the apparent K(d) of initiating GTPs. We propose that D240 and E242 anchor the hairpin to the DNA and position the tip of the hairpin to allow V237 to intercalate and distort the DNA during open complex formation. The interactions of E242 and D240 with the upstream junction of the melted dsDNA promoter also align the template strand within the active site for efficient RNA synthesis.
authors.affiliation: Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
authors.forename: Natalie M
authors.initials: NM
authors.lastname: Stano
authors.forename: Smita S
authors.initials: SS
authors.lastname: Patel
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D013698:Templates, Genetic; D013816:Thermodynamics; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; D012313:RNA; D006160:Guanosine Triphosphate; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases