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

J Mol Biol. 1993 Feb 20;229(4):805-11. doi: 10.1006/jmbi.1993.1086.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriophage T7 / enzymology
  • Bacteriophage T7 / genetics*
  • Base Composition
  • DNA, Viral / metabolism
  • DNA-Directed RNA Polymerases / metabolism*
  • Molecular Sequence Data
  • Mutation
  • Nucleic Acid Conformation
  • Promoter Regions, Genetic*
  • Viral Proteins

Substances

  • DNA, Viral
  • Viral Proteins
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases