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

Dipankar Chatterji, Cheng-Wen Wu Lee, Felicia Y.H. Wu*

*此作品的通信作者

研究成果: Article同行評審

7 引文 斯高帕斯(Scopus)

摘要

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 Förster'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.

原文English
頁(從 - 到)218-225
頁數8
期刊Archives of Biochemistry and Biophysics
244
發行號1
DOIs
出版狀態Published - 1月 1986

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