Escherichia coli RNA polymerase contacts promoter DNA at two upstream regions separated by a spacer DNA. We had previously studied the effects 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 function. We proposed that this effect was due to the fact that the oligo(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 by combining the substitutions with single base pair insertions and deletions in the spacer DNA, which affect the length and the twist in known ways. Deletion and substitutions equally affect the activities of promoters with the presumed B-DNA substitutions. However, for promoters bearing the oligo(dG)-oligo(dC) substitution, a deletion in the spacer DNA impairs promoter activity to a much greater extent than the insertion of a base pair. This asymmetry is consistent with our hypothesis that the deleterious effects of the substitution are due to its having the reduced twist and/or length characteristic of A-DNA. Additionally, we present data that concern the sequence requirements for adoption of this structure that leads to reduced promoter function.