Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor.

Abstract

In enteric bacteria the products of two nitrogen regulatory genes, ntrA and ntrC, activate transcription of ginA, the structural gene encoding glutamine synthetase, both in vivo and in vitro. The ntrC product (gpntrC) is a DNA-binding protein, which binds to five sites in the ginA promoter-regulatory region and appears to activate transcription initiation.Using as an assay the stimulation of ginA transcription in a coupled in vitro transcription-translation system, we have partially purified the ntrA gene product (gpntrA). The following evidence is consistent with the view that gpntrA is a or subunit for RNA polymerase! (i) The gpntrA activity copurifies with the 470 holoenzyme (Eo7O) and core (E) forms of RNA polymerase through several steps but can be separated from them by chromatography on heparin agarose. (ii) After further purification by molecular sieve chromatography, the partially purified gpntrA fraction allows transcription ofginA from the same startpoint used in vivo; transcription is dependent on gpntiC and on added E. The gpntrA fraction does not allow transcription from promoters that we have used as controls, including lacUV5. Ea~70 has the reverse specificity.

DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter.

“…As judged by DNase I-and methylation-protection assays, the NtrC protein binds to five sites in the glnA promoter region (33) (Fig. 1).…”

“…a-54, the NtrC activator protein [NtrCcon mutant form = NtrC610 (9)], and NtrB [the kinase that phosphorylates NtrC (32)] were purified as described (33,34 Proteins were first mixed together and then added to the DNA. For the reactions in Table 1, buffer A was used together with NtrC (360 nM) and wild-type DNA fragment (34 nM).…”

Transformation of Arthrobacter and studies on the transcription of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli

“…It has been demonstrated that Streptomyces, like E. coli (Grossman et al, 1984;Landick et al, 1984), Bacillus (Losick & Pero, 1981) and recently the mntrogen-regulated genes of enteric bacteria (Hirschman et al, 1985) possess multiple sigma (C) factors (Westpheling et al, 1985;Buttner & Brown, 1985) which are each believed to initiate transcription from a specific class of promoter. In S. coelicolor one of the a-factors identified, probably the o.40 found by Buttner & Brown (1985) or o45 of Westpheling et al (1985), may be analogous to the E. coli a`70 factor that recognizes the consensus promoter of TTGACA (-35 region) and TATAAT (-10 region), and may be partly responsible for the capacity of Streptomyces to recognize and initiate transcription from promoters derived from a diverse source of bacteria including E. coli (Bibb & Cohen, 1982;Jaurin & Cohen, 1984;Westpheling et al, 1985;Herbert et al, 1986).…”

The C-terminal RpoN Domain of σ54 Forms an Unpredicted Helix-Turn-Helix Motif Similar to Domains of σ70

“…However, the initiation of transcription at specific DNA sequences requires additional protein(s). In bacteria, these additional proteins are called the " factors" (1,2). The factor facilitates transcription at specific DNA sequences by 1) binding to the core RNAP to form the -RNAP holoenzyme, 2) recognizing and binding to a specific DNA sequence next to the transcription start site, called the promoter element, and 3) opening the double-stranded DNA to initiate transcription.…”

“…The primary protein complex catalyzing transcription is composed of five subunits, ␣ 2 ␤Ј␤, called "core RNA polymerase" (RNAP). 2 Core RNAP is fully competent to synthesize RNA from DNA. However, the initiation of transcription at specific DNA sequences requires additional protein(s).…”