Proximity relationship between the active site of Escherichia coli RNA polymerase and rifampicin binding domain: a resonance energy-transfer study.
Escherichia coli RNA polymerase has two subsites, i and i + 1, for the binding of the first two substrates, and the first phosphodiester bond is formed between them during the initiation of transcription. Various studies have shown earlier that the inhibitor rifampicin has little effect, if any, on the formation of this phosphodiester bond. On an earlier occasion, we measured the distance of the i nucleotide from the rifampicin binding site on RNA polymerase using Forster's energy-transfer mechanism [Kumar & Chatterji (1990) Biochemistry 29,317]. In this paper, the 1-aminonaphthalene-5-sulfonic acid (AmNS) derivative of UTP in the presence of 10 mM MgCl2 was used as an energy donor, and its distance from rifampicin was estimated. The modified nucleotide (gamma-AmNS)-UTP binds to RNA polymerase with a Kd of 3 microM and has one binding site in the presence of Mg(II) ion. Fluorescence titration studies performed with or without an initiator indicated that (gamma-AmNS)-UTP exclusively binds to RNA polymerase at the (i + 1) site in the presence of Mg(II). Rifampicin was found to form a 1:1 complex with RNA polymerase bound to labeled UTP. Rifampicin and (gamma-AmNS)-UTP have a substantial spectral overlap with an energy-transfer efficiency close to 50%. Labeled UTP shows a decrease in its excited-state lifetime when bound to the enzyme; the transfer efficiency calculated from lifetime measurements was found to be lower than that estimated from steady-state spectral analysis. Time-resolved emission spectral analysis was carried out to differentiate between the free and bound UTP over the enzyme surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Kumar
K P
KP
Centre for Cellular and Molecular Biology, Hyderabad, India.
Reddy
P S
PS
Chatterji
D
D
D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D008433:Mathematics; D008961:Models, Structural; D008962:Models, Theoretical; D009282:Naphthalenesulfonates / Q000138:chemical synthesis / Q000378:metabolism*; D011487:Protein Conformation; D012293:Rifampin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C027081:UTP-1-aminonaphthalene-5-sulfonate; D014364:Tryptophan; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin