Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase.
Terbium (III) upon complexation with guanosine 5'-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm. Analysis of the binding data yielded a value for the mean Kd between Tb(III) and GTP of 0.2 microM, with three binding sites for Tb(III) on GTP. 31P and 1H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP. Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a Kd value of 4 microM between the TbGTP and the enzyme. It was observed that TbGTP can be incorporated in the place of GTP during E. coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter. Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the beta-subunit of E. coli RNA polymerase. This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin. Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 A for the 488- and 545-nm emission bands, respectively. The distance between the substrate binding site and the rifampicin binding site on the beta-subunit of E. coli RNA polymerase was measured to be around 30 A. This suggests that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate.
Centre for Cellular and Molecular Biology, Andhra Pradesh, India.
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KP
Kumar
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Chatterji
D001665:Binding Sites; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D010710:Phosphates / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012293:Rifampin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013604:T-Phages / Q000235:genetics; D013725:Terbium / Q000378:metabolism / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D010710:Phosphates; D013725:Terbium; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin