HO. and DNase I probing of E sigma 70 RNA polymerase--lambda PR 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 complexes RPo1 (-Mg2+) and RPo2 (+Mg2+), formed by Escherichia coli RNA polymerase (E sigma 70) at the lambda PR promoter. We report that HO. detects major differences in backbone reactivity between RPo1 and RPo2 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. in RPo2 but are relatively protected in RPo1. Binding of Mg2+ to convert RPo1 to RPo2 therefore increases the reactivity of two negatively charged footprinting agents [MnO4-; Suh, W.-C., Ross, W., & Record, M. T., Jr., (1993) Science 259, 358-361; and Fe(EDTA)2-/HO.] 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 Mg2+ 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 RPo1 and RPo2 is continuously protected from DNase I and hydroxyl radical (HO.) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D018983:DNA Footprinting; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I; D017665:Hydroxyl Radical; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000378:metabolism*; D014158:Transcription, Genetic
D004279:DNA, Viral; D012808:Sigma Factor; D017665:Hydroxyl Radical; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D008274:Magnesium