Hierarchy of base-pair preference in the binding domain of the bacteriophage T7 promoter

The activity of bacteriophage T7 RNA polymerase (RNAP) at a collection of T7 promoter mutants having all possible single base-pair substitutions in the region from -15 to -6 was determined by transcription in vitro, thus establishing a hierarchy of base-pair preference at each position. The tolerance of the RNAP for base-pair substitutions is not uniform across the binding domain. Under stringent conditions (20 mM-MgCl2), T7 RNAP is highly permissive for all base-pair substitutions at -13 and -12. The RNAP is partially permissive at -15, -14, -11, -10 and -6, and exhibits a clear pattern of base-pair preference at these positions. The RNAP is non-permissive for substitutions at -9 to -7, and will accept only the consensus base-pairs at these positions. Under lower stringency conditions (8 mM-MgCl2, or additionally in the presence of dimethylsulfoxide) a decrease in specificity is observed at most positions except -9. Analysis of these data suggests potential contacts that may be important for promoter function.