Intrinsic fluorescence of E. coli RNA polymerase as a probe for its conformational changes during transcription initiation.
A simple fluorimetric assay based on internal fluorescence of tryptophan residues of E. Coli RNA polymerase has been developed to ascertain the number of steps during conversion of closed complex of the polymerase-promoter (trp promoter cloned in plasmid pDR720) to open complex. Our results from measurement on relative ratio of fluorescence at 340 nm (lambda ex = 295 nm) for free and promoter-bound RNA polymerase as a function of temperature, within the range 4 degrees C to 37 degrees C, indicate following equilibria for the above conversion: R+P<-->RPc<-->RPi1<-->RPi2<-->RPo. Apart from detection of one more intermediate in terms of conformational states of the bound RNA polymerase, second feature of our studies is the examination of conformational state of the polymerase using accessibility of fluorophor, tryptophan residues, to a neutral quencher, acrylamide, as the probe. We observe that in terms of accessibility of tryptophan residues in protein, intermediate complex, RPi2, is conformationally most perturbed in comparison to free polymerase. Implications of these results are discussed and compared with the available reports from footprinting and gel retardation assays of RNA polymerase-promoter interactions.
Sen
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Biophysics Division, Saha Institute of Nuclear Physics, Calcutta, India.
Dasgupta
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D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013816:Thermodynamics; D014158:Transcription, Genetic; D014364:Tryptophan
D014364:Tryptophan; D012321:DNA-Directed RNA Polymerases