Construction of bacteriophage T7 late promoters with point mutations and characterization by in vitro transcription properties

Abstract

This paper describes the construction of 18 cloned bacteriophage T7 late promoters with single point mutations. In vitro transcription experiments were used to characterize the properties of these promoters. Since the mutated promoters are cloned into identical backgrounds, differences seen in the transcription assays are directly attributable to the point mutations. All of the mutated promoters are less active than wildtype, but they can be divided into two types. Type A mutations map from -4 to +1 and reduce promoter activity when the template is linearized or when 60mM NaCl is added to the reaction buffer. Type B mutations map from -9 to -7 and reduce promoter activity under all conditions tested. At several sites all three possible point mutations are available. At these sites we observed hierarchies of base pair preference, as determined by promoter activity, that may indicate that T7 RNA polymerase interacts with groups in the major groove.