The bacterial TATAAT −10 region sequence was the first promoter element to be identified, but how it functions is still not clear. Because the duplex element is melted during initiation, the effects of substitutions were studied in both single-and double-strand contexts. Band-shift results were particularly unexpected in the context of melted DNA. The effect of the lac UV5-melted −10 region on polymerase binding was found to include a large sequence nonspecific contribution. Instead the dominant role of single-stranded −10 region nucleotides was in directing the isomerization of the RNA polymerase to its heparin resistant form. This role becomes minimal when the melting is extended beyond the −10 region to encompass the transcription start site, as in the final open complex. The duplex binding results are in agreement with previous reports that showed positions −12T and −11A are of primary importance for promoter recognition. Thus the consensus −10 region sequences function in two ways, both before full promoter melting. They stabilize initial polymerase binding via duplex interactions and subsequently as single-stranded DNA they promote enzyme isomerization to the functional form.