Mechanism of regulation of transcription initiation by ppGpp. II. Models for positive control based on properties of RNAP mutants and competition forRNAP

Strains containing ppGpp, a nucleotide whose synthesis is dependent on the RelA and SpoT proteins of Eschericlzia coil, display slightly lower rRNA pr omoter activity and much higher amino acid biosynthesis/transport promoter activity than Delta rel Delta spoT strains. In the accompanying paper, we s how that ppGpp directly inhibits rRNA promoter activity in vitro by decreas ing the lifetime of the a rrn P1 open complex. However, ppGpp does not stim ulate amino acid promoter activity in vitro. We show here that RNA polymera se (RNAP) mutants, selected to confer prototrophy to al Delta rel Delta spo T strains, mimic the effects of ppGpp on wild-type RNAP. Based on the posit ions of the mutant residues that confer prototrophy in the structure of cor e RNAP, we suggest molecular models for how the mutants, and by analogy ppG pp, generally decrease the lifetime of open complexes. We show that amino a cid promoters require higher concentrations of RNAP for function in vitro a nd in vivo than control promoters, and are more sensitive to competition fo r RNAP in vivo than control promoters. Furthermore, we show that the requir ement of an amino acid promoter for ppGpp in vivo can be alleviated by incr easing its rate-limiting RNAP-binding step. Our data are consistent with a previously proposed passive model in which ppGpp inhibits stable RNA synthe sis directly by reducing the Lifetime of the rrn P1 open complex, Liberatin g enough RNAP to stimulate transcription from amino acid promoters. Our dat a also place considerable constraints on models invoking hypothetical facto rs that might increase amino acid promoter activity in a ppGpp-dependent fa shion. (C) 2001 Academic Press.