Specificity of promoter utilization in bacterial RNA polymerases is imparted by a class of proteins referred to as sigma factors. Conserved region 2.3 of these proteins is thought to play a role in the strand separation process that occurs during the formation of an initiation-competent RNA polymerase-promoter complex. We have used a heterologous system consisting of Escherichia coli core RNA polymerase and Bacillus subtilis sigma A to probe the effects of amino acid substitutions in region 2.3. In agreement with previous work [Juang & Helmann (1994) J. Mol. Biol. 235, 1470-1488] we observe that several amino acid substitutions exacerbate the deleterious effect of low temperature on promoter-dependent initiation. On the other hand, no such enhanced cold sensitivity is found with double-stranded templates that contain short "bubbles" of single-stranded DNA, indicating that the DNA-melting defect imposed by these mutant sigma factors can be suppressed by the use of such bubble templates. These results support the involvement of region 2.3 in the strand separation process that accompanies open complex formation at promoters.