Site-directed mutagenesis of the -10 region of the lacUV5 promoter. Introduction of dA4.dT4 tract suppresses open complex formation.

Abstract

Homopolymeric dAn.dTn sequences, where n is 4 or greater, have special properties leading to increased duplex stability and DNA bending. The lacUV5 promoter was used to examine the functional consequences of changing the -10 TATAAT consensus sequence to the sequence TAAAAT. The transversion mutation at the underlined site was accomplished with site-directed mutagenesis using translation termination as the selection procedure. For free DNA, structural differences at the 5' and 3' junction regions of the dA4.dT4 tract can be readily detected by DNase I digestion. However, site binding by Escherichia coli RNA polymerase appeared unaltered by the TAAAAT sequence since identical DNase I footprints were obtained for the lacUV5 and mutant promoters. Binding competition studies under different ionic strengths revealed a significant reduction in mutant promoter open complex formation relative to the lacUV5 promoter. Mutant promoter open complexes also dissociated faster and to a greater extent than the corresponding lacUV5 promoter open complexes when challenged with heparin or a combination of heparin and increased KCl concentration. Consequently, mutant promoter open complexes appear less stable than lacUV5 promoter open complexes.