Substantial effort has been made to investigate the interactions that the Escherichia coli RNA polymerase makes with promoter DNA during transcription initiation. The lacUV5 promoter has been the object of many of these studies, and to date, an incredible wealth of information exists on how RNA polymerase interacts with this promoter. We have sought to expand current knowledge by the use of two chemical interference protocols, phosphate ethylation and missing nucleoside. We have added to existing information with the identification of additional phosphates, for example, at the start site of the template strand that, when ethylated, perturb the binding of RNA polymerase. We have also discovered a number of positions, most remarkably -37 to -34 of the nontemplate strand, where nucleoside loss decreases binding. Finally, we have discovered positions of ethylation and/or nucleoside loss that can stimulate binding. In particular, missing nucleosides and phosphate ethylation near the transcription start site enhance RNA polymerase binding.