In transcription initiation, the DNA strands must be separated to expose the template to RNA polymerase. As the closed initiation complex is converted to an open one, specific protein-DNA interactions involving bases of the nontemplate strand form and stabilize the promoter complex in the region of unwinding. Specific interaction between RNA polymerase and the promoter in Escherichia coli was detected and quantified as the binding affinity of nontemplate oligonucleotide sequences. The RNA polymerase subunit sigma factor 70 contacted the bases of the nontemplate DNA strand through its conserved region 2; a mutation that affected promoter function altered the binding affinity of the oligonucleotide to the enzyme.