Structural studies of lacUV5-RNA polymerase interactions in vitro - Ethylation interference and missing nucleoside analysis

Substantial effort has been made to investigate the interactions that the E scherichia coli RNA polymerase makes with promoter DNA during transcription initiation. The lacUV5 promoter has been the object of many of these studi es, and to date, an incredible wealth of information exists on how RNA poly merase interacts with this promoter. We have sought to expand current knowl edge by the use of two chemical interference protocols, phosphate ethylatio n and missing nucleoside. We have added to existing information with the id entification of additional phosphates, for example, at the start site of th e template strand Chat, when ethylated, perturb the binding of RNA polymera se. We have also discovered a number of positions, most remarkably -37 to - 34 of the nontemplate strand, where nucleoside loss decreases binding. Fina lly, we have discovered positions of ethylation and/or nucleoside loss that can stimulate binding. In particular, missing nucleosides and phosphate et hylation near the transcription start site enhance RNA polymerase binding.