GUANOSINE TETRAPHOSPHATE-INDUCED DISSOCIATION OF OPEN COMPLEXES AT THE ESCHERICHIA-COLI RIBOSOMAL-PROTEIN PROMOTERS RPLJ AND RPSA P1 - NANOSECOND DEPOLARIZATION SPECTROSCOPIC STUDIES

We have measured the fluorescence anisotropy decays of various transcr iption complexes formed between Escherichia coli RNA polymerase (RNAP) and the rplJ, rpsA P1 and lacUV5 promoters, where the sigma(70)-subun it of RNAP is covalently labeled with the fluorescent probe 1,5-IAEDAN S. The observed changes in the rotational correlation times (phi(r)) o f the sigma(70)-bound probe upon ppGpp or NTP addition to preformed op en 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 a nd nucleotides), results in the dissociation of RNAP from the binary c omplex. 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 protei n promoters. One is transcription-competent and releases sigma(70) upo n elongation, while the other dissociates from the open complex. A fra ction of the latter species releases the sigma(70) subunit and is unab le to form a transcription-competent holoenzyme. Our data supports the mechanism of open complex-destabilization at stringent promoters by p pGpp. (C) 1998 Elsevier Science B.V. All rights reserved.