HO times and DNase I probing of E sigma super(70) RNA polymerase- lambda P sub(R) promoter open complexes: Mg super(2+) binding and its structural consequences at the transcription start site

• Published in: Biochemistry (Easton) Vol. 34; no. 48; pp. 15624 - 15632
• Main Authors: Craig, M L, Suh, Won-Chul, Record, M T
• Format: Journal Article
• Language: English
• Published: 01.01.1995
• Subjects: Escherichia coli
• ISSN: 0006-2960

Chemical and enzymatic probing (footprinting) of the reactivity of the promoter DNA backbone is applied to characterize two binary open complexes, RP sub(o1) (-Mg super(2+)) and RP sub(o2) (+Mg super(2+)), formed by Escherichia coli RNA polymerase (E sigma super(70)) at the lambda P sub(R) promoter. We report that HO times detects major differences in backbone reactivity between RP sub(o1) and RP sub(o2) in the open region from -4 to +1 relative to the transcription start site. Deoxyribose sugars at positions -4 to +1 of the t (template) strand react with HO times in RP sub(o2) but are relatively protected in RP sub(o1). Binding of Mg super(2+) to convert RP sub(o1) to RP sub(o2) therefore increases the reactivity of two negatively charged footprinting agents at the start site and is required for binding of the negatively-charged initiating nucleotides to the polymerase and the t strand at the start site. We propose that these effects result from binding of two Mg super(2+) ions to the catalytic carboxyls in the nucleotide binding sites. Except for the key region on the t strand at the start site, the promoter DNA of both RP sub(o1) and RP sub(o2) is continuously protected from DNase I and hydroxyl radical (HO times ) cleavage between the -12 and +25 promoter positions. Protection in the upstream region, extending from -13 to about -70, is periodic, with an 11 base pair periodicity indicative of binding of polymerase to a single face of the DNA helix. Since the backbone of approximately 6-7 of every 11 base pairs are protected, binding may involve a relatively deep surface groove of E sigma super(70). Major enhancements of HO times and DNase I cleavage at -38 and -48 suggest bending-induced distortion of the double helix, consistent with a model in which the upstream region of the promoter DNA is wrapped around E sigma super(70). Mg super(2+) binding in the RP sub(o1) arrow right RP sub(o2) conversion causes changes in the extent of the HO times enhancements. Structural models for the two open complexes at lambda P sub(R) are discussed and compared in the context of RP sub(o2) (+Mg super(2+)) at different promoters.