Guanosine tetraphosphate-induced dissociation of open complexes at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1: nanosecond depolarization spectroscopic studies

Biophys Chem. 1998 Oct 5;75(1):21-32. doi: 10.1016/s0301-4622(98)00186-0.

Abstract

We have measured the fluorescence anisotropy decays of various transcription complexes formed between Escherichia coli RNA polymerase (RNAP) and the rplJ, rpsA P1 and lacUV5 promoters, where the sigma 70-subunit of RNAP is covalently labeled with the fluorescent probe 1,5-IAEDANS. The observed changes in the rotational correlation times (phi r) of the sigma 70-bound probe upon ppGpp or NTP addition to preformed open complexes, were used to directly infer the extent of association of the sigma-subunit with these transcription complexes. At the rplJ and rpsA P1 promoters, the addition of ppGpp (in the absence of heparin and nucleotides), results in the dissociation of RNAP from the binary complex. This is either accompanied by, or leads to the dissociation of a fraction of the holoenzyme-bound sigma 70. At the lacUV5 promoter, only a marginal dissociation of RNAP is observed. We propose a model where two types of ppGpp-bound RNAP interact with the ribosomal protein promoters. One is transcription-competent and releases sigma 70 upon elongation, while the other dissociates from the open complex. A fraction of the latter species releases the sigma 70 subunit and is unable to form a transcription-competent holoenzyme. Our data supports the mechanism of open complex-destabilization at stringent promoters by ppGpp.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anisotropy
  • Bacterial Proteins / metabolism*
  • DNA-Directed DNA Polymerase / metabolism
  • DNA-Directed RNA Polymerases / metabolism
  • Escherichia coli / drug effects
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Fluorescent Dyes
  • Guanosine Tetraphosphate / pharmacology*
  • Naphthalenesulfonates
  • Ribosomal Protein L10
  • Ribosomal Proteins / metabolism*
  • Spectrophotometry, Ultraviolet
  • Transcription, Genetic

Substances

  • Bacterial Proteins
  • Fluorescent Dyes
  • JofD protein, Bacillus subtilis
  • Naphthalenesulfonates
  • Ribosomal Proteins
  • Guanosine Tetraphosphate
  • 1,5-I-AEDANS
  • DNA-Directed RNA Polymerases
  • DNA-Directed DNA Polymerase