Evidence for a ppGpp-binding site on Escherichia coli RNA polymerase: proximity relationship with the rifampicin-binding domain.
On amino acid starvation, Escherichia coli cells exhibit an adaptive facility termed the stringent response. This is characterized by the production of high levels of a regulatory nucleotide, ppGpp, and concomitant curtailment in rRNA synthesis. Various studies reported earlier indicated that RNA polymerase is the site of action of ppGpp although a direct demonstration of the interaction of ppGpp with E. coli RNA polymerase is still lacking. Here we report the labelling of ppGpp with a fluorescent probe, 1-aminonapthalene-5-sulphonate (AmNS), at the terminal phosphates. AmNS-ppGpp responded much like a ppGpp molecule in an in vitro total transcription assay at selective promoters. Fluorescence titration of the tryptophan emission of RNA polymerase by AmNS-ppGpp indicated a unique binding site in the absence of template DNA. Competition experiments showed that unlabelled ppGpp binds to the enzyme at the same site. Sigma factor seems to have no effect on this binding. The titration profile is also characterized by a single slope in the Scatchard analysis. The presence of GTP or GDP does not influence the binding of AmNS-ppGpp with RNA polymerase. Forster's distance measurement was carried out which placed AmNS-ppGpp 27 A away from the rifampicin-binding domain of RNA polymerase.
Reddy
P S
PS
Centre for Cellular and Molecular Biology, Hyderabad, India.
Raghavan
A
A
Chatterji
D
D
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D015964:Gene Expression Regulation, Bacterial; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006159:Guanosine Tetraphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D009282:Naphthalenesulfonates; D011485:Protein Binding / Q000187:drug effects; D012318:RNA Polymerase I / Q000378:metabolism*; D012293:Rifampin / Q000378:metabolism*; D012808:Sigma Factor / Q000378:metabolism
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D012808:Sigma Factor; D006153:Guanosine Diphosphate; D006159:Guanosine Tetraphosphate; D006160:Guanosine Triphosphate; D012318:RNA Polymerase I; D012293:Rifampin; C022042:1-naphthylamine-5-sulfonic acid