PROMOTER RECOGNITION BY ESCHERICHIA-COLI RNA-POLYMERASE - EFFECTS OF SINGLE-BASE PAIR DELETIONS AND INSERTIONS IN THE SPACER DNA SEPARATINGTHE -10 AND -35 REGIONS ARE DEPENDENT ON SPACER DNA-SEQUENCE

Escherichia coli RNA polymerase contacts promoter DNA at two upstream regions separated by a spacer DNA. We had previously studied the effec ts of substitutions of simple DNA sequences in a stretch of the spacer DNA devoid of any known specific contacts with RNA polymerase. It was found that substitution of nine consecutive nonalternating dG-dC base pairs, but not nine alternating dG-dC base pairs, impaired promoter f unction. We proposed that this effect was due to the fact that the oli go(dG)-oligo(dC) sequence adopted a conformation (possibly A-helical) resulting in a reduction in its length and twist as compared with the B-form DNA of the alternating sequence. Here we test this hypothesis b y combining the substitutions with single base pair insertions and del etions in the spacer DNA, which affect the length and the twist in kno wn ways. Deletions and substitutions equally affect the activities of promoters with the presumed B-DNA substitutions. However, for promoter s bearing the oligo(dG)-oligo(dC) substitution, a deletion in the spac er DNA impairs promoter activity to a much greater extent than the ins ertion of a base pair. This asymmetry is consistent with our hypothesi s that the deleterious effects of the substitution are due to its havi ng the reduced twist and/or length characteristic of A-DNA. Additional ly, we present data that concern the sequence requirements for adoptio n of this structure that leads to reduced promoter function.