Role of T7 RNA polymerase His784 in start site selection and initial transcription.
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.
Brieba
Luis G
LG
Department of Biochemistry, University of Texas Health Sciences Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760, USA.
Padilla
Robert
R
Sousa
Rui
R
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
D014764:Viral Proteins; D005973:Glutamine; D006151:Guanosine; D006639:Histidine; D006160:Guanosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000409:Alanine