The molecular topography of RNA polymerase-promoter interaction

Ultraviolet irradiation forms covalent crosslinks between E. coli RNA polymerase and the lac UV5 promoter substituted with bromouracil in the place of thymine. I have determined the polymerase subunit and the base within the promoter sequence that are joined to each other in two such crosslinks. The sigma and beta subunits of RNA polymerase, respectively, are crosslinked to the third base upstream (-3) and the second base downstream (+3) from the starting point of transcription (+1). Both bases are on the nontemplate strand of the promoter DNA. The location of the beta subunit suggests that it forms at least part of the catalytic site of the enzyme. The disposition of sigma suggests that this subunit plays a direct role in unwinding the DNA at the promoter. The sigma crosslink is close to the "Pribnow Box," which is centered about 10 bases upstream from the RNA start site, contains a striking homology between promoters and is the locus of many promoter mutations.