HO-CENTER-DOT AND DNASE-I PROBING OF E-SIGMA(70) RNA POLYMERASE-LAMBDA-P-R PROMOTER OPEN COMPLEXES - MG2+ BINDING AND ITS STRUCTURAL CONSEQUENCES AT THE TRANSCRIPTION START SITE

Chemical and enzymatic probing (footprinting) of the reactivity of the promoter DNA backbone is applied to characterize two binary open comp lexes, RP(o1) (-Mg2+) and RP(o2) (+Mg2+), formed by Escherichia coli R NA polymerase (E sigma(70)) at the lambda P-R promoter. We report that HO . detects major differences in backbone reactivity between RP(o1) and RP(o2) in the open region from -4 to +1 relative to the transcript ion start site. Deoxyribose sugars at positions -4 to +1 of the t (tem plate) strand react with HO . in RP(o2) but are relatively protected i n RP(o1). Binding of Mg2+ to convert RP(o1) to RP(o2) therefore increa ses the reactivity of two negatively charged footprinting agents [MnO4 -; Suh, W.-C., Ross, W., & Record, M, T., Jr., (1993) Science 259, 358 -351; and Fe(EDTA)(2-)/HO .] at the start site and is required for bin ding of the negatively-charged initiating nucleotides to the polymeras e and the 1 strand at the start site, We propose that these effects re sult from binding of two Mg2+ ions to the catalytic carboxyls in the n ucleotide binding sites. Except for the key region on the 1 strand at the start site, the promoter DNA of both RP(o1) and RP(o2) is continuo usly protected from DNase I and hydroxyl radical (HO .) cleavage betwe en the -12 and +25 promoter positions, Protection in the upstream regi on, extending from -13 to about -70, is periodic, with an 11 base pair periodicity indicative of binding of polymerase to a single face of t he DNA helix, Since the backbone of approximately 6-7 of every 11 base pairs are protected, binding may involve a relatively deep surface gr oove of E sigma(70). Major enhancements of HO . and DNase I cleavage a t -38 and -48 suggest bending-induced distortion of the double helix, consistent with a model in which the upstream region of the promoter D NA is wrapped around E sigma 70. Mg2+ binding in the RP(o1) --> RP(o2) conversion causes changes in the extent of the HO . enhancements. Str uctural models for the two open complexes at lambda P-R are discussed and compared in the context of RP(o2) (+Mg2+) at different promoters.