title: The transition to an elongation complex by T7 RNA polymerase is a multistep process.
abstract: During the transition from an initiation complex to an elongation complex (EC), T7 RNA polymerase undergoes major conformational changes that involve reorientation of a "core" subdomain as a rigid body and extensive refolding of other elements in the 266 residue N-terminal domain. The pathway and timing of these events is poorly understood. To examine this, we introduced proline residues into regions of the N-terminal domain that become alpha-helical during the reorganization and changed the charge of a key residue that interacts with the RNA:DNA hybrid 5 bp upstream of the active site in the EC but not in the initiation complex. These alterations resulted in a diminished ability to make products >5-7 nt and/or a slow transition through this point. The results indicate that the transition to an EC is a multistep process and that the movement of the core subdomain and reorganization of certain elements in the N-terminal domain commence prior to promoter release (at 8-9 nt).
authors.affiliation: Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
authors.forename: Rajiv P
authors.initials: RP
authors.lastname: Bandwar
authors.forename: Na
authors.initials: N
authors.lastname: Ma
authors.forename: Steven A
authors.initials: SA
authors.lastname: Emanuel
authors.forename: Michael
authors.initials: M
authors.lastname: Anikin
authors.forename: Dmitry G
authors.initials: DG
authors.lastname: Vassylyev
authors.forename: Smita S
authors.initials: SS
authors.lastname: Patel
authors.forename: William T
authors.initials: WT
authors.lastname: McAllister
mesh_terms: D017123:Bacteriophage T7 / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D014764:Viral Proteins; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin