Spatial relationship between the intrinsic metal in the beta subunit and cysteine-132 in the sigma subunit of Escherichia coli RNA polymerase: a resonance energy transfer study.
Fluorescence excited-state energy transfer measurements were carried out between the N-(1-pyrene)maleimide (PM)-labeled sigma subunit and Co in the beta subunit of Co-Zn RNA polymerase (RPase). sigma subunit with or without PM labeling was cleaved with 2-nitro-5-thiocyanobenzoic acid, and the reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One molecule of the fluorescent probe (PM) was found to be attached to the cysteine-132 residue of the sigma subunit. When excited at 340 nm, the fluorescence emission bands from 380 to 420 nm of PM-labeled sigma overlap with the charge transfer absorption band of Co-Zn RPase around 400 nm. Based on Förster's equation, the R0 values for the donor-acceptor pair were calculated to be 21.5 and 22 A in the absence and presence of template analog (dA-dT)60, respectively. Using these R0 values and the observed energy transfer efficiencies, the distance between the cysteine-132 of the sigma subunit and Co located at the initiation site of the beta subunit was calculated to be 22 A with or without the template present, indicating that no major conformational change of the enzyme was induced upon template binding. However, a small but significant change in the above distance was observed upon the addition of ATP to RPase in the presence (dA-dT)60 but not in the absence of (dA-dT)60 template. The biological implications of these observations are discussed.
D001665:Binding Sites; D003545:Cysteine / Q000032:analysis*; D012321:DNA-Directed RNA Polymerases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D008301:Maleimides; D008433:Mathematics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013698:Templates, Genetic; D015032:Zinc / Q000032:analysis*
D008301:Maleimides; C003944:N-(3-pyrene)maleimide; D012321:DNA-Directed RNA Polymerases; D015032:Zinc; D003545:Cysteine