Spatial relationship between the intrinsic metal in the β subunit and cysteine-132 in the σ subunit of Escherichia coli RNA polymerase: a resonance energy transfer study

Abstract

Fluorescence excited-state energy transfer measurements were carried out between the N-(1-pyrene)maleimide (PM)-labeled σ subunit and Co in the β subunit of Co-Zn RNA polymerase (RPase). σ 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 σ subunit. When excited at 340 nm, the fluorescence emission bands from 380 to 420 nm of PM-labeled σ overlap with the charge transfer absorption band of Co-Zn RPase around 400 nm. Based on Forster's equation, the R₀ values for the donor-acceptor pair were calculated to be 21.5 and 22 Å 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 σ subunit and Co located at the initiation site of the β subunit was calculated to be 22 Å 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.