EVIDENCE FOR A PPGPP-BINDING SITE ON ESCHERICHIA-COLI RNA-POLYMERASE - PROXIMITY RELATIONSHIP WITH THE RIFAMPICIN-BINDING DOMAIN

On amino acid starvation, Escherichia coil cells exhibit an adaptive f acility termed the stringent response. This is characterized by the pr oduction of high levels of a regulatory nucleotide, ppGpp, and concomi tant curtailment in rRNA synthesis. Various studies reported earlier i ndicated that RNA polymerase is the site of action of ppGpp although a direct demonstration of the interaction of ppGpp with E. coil RNA pol ymerase is still lacking. Here we report the labelling of ppGpp with a fluorescent probe, 1-aminonapthalene-5-sulphonate (AmNS), at the term inal phosphates. AmNS-ppGpp responded much like a ppGpp molecule in an in vitro total transcription assay at selective promoters. Fluorescen ce titration of the tryptophan emission of RNA polymerase by AmNS-ppGp p indicated a unique binding site in the absence of template DNA. Comp etition experiments showed that unlabelled ppGpp binds to the enzyme a t 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 measurem ent was carried out which placed AmNS-ppGpp 27 Angstrom away from the rifampicin-binding domain of RNA polymerase.