title: Exposure of T7 RNA polymerase to the isolated binding region of the promoter allows transcription from a single-stranded template.
abstract: While the binding region of the T7 promoter must be double-stranded (ds) to function, the non-template strand in the initiation region is dispensable, and a promoter that lacks this element allows efficient initiation. To determine whether the binding region serves merely to recruit the RNA polymerase (RNAP) to the vicinity of a melted initiation region or provides other functions, we utilized a GAL4-T7 RNAP fusion protein to provide an independent binding capacity to the RNAP. When the GAL4-T7 RNAP was recruited to a single-stranded (ss) promoter via a nearby Gal4 recognition sequence, no transcription was observed. However, transcription from the ss promoter could be activated by the addition, in trans, of a ds hairpin loop that contains only the binding region of the promoter. The same results were obtained in the absence of the GAL4 recognition sequence in the template and were also observed with wild type enzyme. Gel-shift experiments indicate that exposure of the RNAP to the isolated binding region facilitates recruitment of the ss template, but that the binding region is displaced from the complex prior to initiation. We conclude that exposure of the RNAP to the isolated binding region reorganizes the enzyme, allowing it to bind to the ss template. These findings have potential implications with regard to mechanisms of promoter binding and melting.
authors.affiliation: Morse Institute of Molecular Genetics, Department of Microbiology and Immunology, SUNY Downstate Medical Center, Brooklyn, New York 11203, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kukarin
authors.forename: Minqing
authors.initials: M
authors.lastname: Rong
authors.forename: William T
authors.initials: WT
authors.lastname: McAllister
mesh_terms: D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013816:Thermodynamics; D014158:Transcription, Genetic; D014764:Viral Proteins
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases