Transcription from bacteriophage T7 and SP6 RNA polymerase promoters in the presence of 3'-deoxyribonucleoside 5'-triphosphate chain terminators

Abstract

RNA synthesis by T7 RNA polymerase or SP6 RNA polymerase is 100-1000 times more sensitive to the presence of the 3''-deoxyribonucleoside 5''-triphosphate chain terminators than is RNA synthesis by Escherichia coli RNA polymerase or Q.beta. replicase. These ribonucleotide analogues do not alter the specificity of each polymerase for its own promoters nor do they alter the site at which synthesis is initiated. Transcription by T7 RNA polymerase or SP6 RNA polymerase in the presence of relatively low concentrations of these chain terminators offers a useful route for determining the nucleotide sequence of any DNA segment that is inserted immediately downstream from a homologous bacteriophage promoter. This sequencing procedure was used to explore the effects that different dinucleotides have on the specificity of initiation at two different T7 RNA polymerase promoters.