title: New insights into the mechanism of initial transcription: the T7 RNA polymerase mutant P266L transitions to elongation at longer RNA lengths than wild type.
abstract: RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially, transcribing complexes are characteristically unstable, yielding short abortive products on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e. at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multisubunit RNA polymerases are discussed.
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
authors.forename: Luis E
authors.initials: LE
authors.lastname: Ramírez-Tapia
authors.forename: Craig T
authors.initials: CT
authors.lastname: Martin
mesh_terms: D019943:Amino Acid Substitution; D017123:Bacteriophage T7 / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics; D005454:Fluorescence Polarization; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D059748:Proteolysis; D014158:Transcription, Genetic; D014357:Trypsin / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin