title: Role of T7 RNA polymerase His784 in start site selection and initial transcription.
abstract: The role of steric constraints vs sequence preference in start site selection by T7 RNA polymerase was investigated by using a series of synthetic promoters in which the preferred template strand 'CC' initiation sequence was moved away from its normal position relative to the -17 to -6 element of the T7 promoter. It was found that the CC sequence directs efficient initiation if placed 1 or 2 nt downstream of its normal position, but not if placed upstream, or more than 2 nt downstream, of +1. Mutagenesis revealed that part of the bias to initiate with GTP is due to an interaction between histidine 784 and the 2-amino group of a guanosine bound in the initiating triphosphate position. This interaction is also important for holding short transcripts within the transcription complex during initial transcription.
authors.affiliation: Department of Biochemistry, University of Texas Health Sciences Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760, USA.
authors.forename: Luis G
authors.initials: LG
authors.lastname: Brieba
authors.forename: Robert
authors.initials: R
authors.lastname: Padilla
authors.forename: Rui
authors.initials: R
authors.lastname: Sousa
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics*; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D006151:Guanosine / Q000737:chemistry / Q000235:genetics; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics*; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D013237:Stereoisomerism; D024363:Transcription Initiation Site; D014764:Viral Proteins
chemical_list: D014764:Viral Proteins; D005973:Glutamine; D006151:Guanosine; D006639:Histidine; D006160:Guanosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000409:Alanine