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The nitrogen regulatory (NtrC) protein of enteric bacteria, which binds to sites that have the properties of transcriptional enhancers, is known to activate transcription by a form of RNA polymerase that contains the NtrA protein (sigma 54) as sigma factor (referred to as sigma 54-holoenzyme). In the presence of adenosine triphosphate, the NtrC protein catalyzes isomerization of closed recognition complexes between sigma 54-holoenzyme and the glnA promoter to open complexes in which DNA in the region of the transcription start site is locally denatured. NtrC is not required subsequently for maintenance of open complexes or initiation of transcription.

The NTRC protein (ntrC product) of enteric bacteria activates transcription of nitrogen-regulated genes by a holoenzyme form of RNA polymerase that contains the ntrA product (oa') as a factor. Although unmodified NTRC will bind to DNA, it must be phosphorylated to activate transcription. Both phosphorylation and dephosphorylation of NTRC occur in the presence of the NTRB protein (ntrB product). We here demonstrate rigorously that it is the NTRB protein that is a protein kinase by showing that NTRB can phosphorylate itself, whereas NTRC cannot. Phosphorylated NTRC (NTRC-P) is capable of autodephosphorylation with a first-order rate constant of 0.14-0.19 min-' (t112 of 5.0-3.6 min) at 3rC. In addition, there is regulated dephosphorylation of NTRC-P. By contrast to the autophosphatase activity, regulated dephosphorylation requires three components in addition to NTRC-P: the PI, regulatory protein, NTRB, and ATP. NTRC is phosphorylated within its amino-terminal domain, which is conserved in one partner ofa number of two-component regulatory systems in a wide variety of eubacteria. A purified aminoterminal fragment of NTRC (-12.5 kDa) is sufficient for recognition by NTRB and is autodephosphorylated at the same rate as the native protein.Together with a holoenzyme form of RNA polymerase containing the ntrA product (a"4) as o-factor, the NTRC protein (ntrC product) of enteric bacteria activates transcription of a number of genes in response to availability of combined nitrogen (refs. 1-3, see ref. 4 for review). Ninfa and Magasanik discovered that activation of transcription was correlated with phosphorylation of NTRC, which occurred in the presence of the NTRB protein (ntrB product) and ATP (5). Consistent with genetic studies, preliminary biochemical studies indicated that the balance between phosphorylated and unphosphorylated forms of NTRC was controlled by the PI, regulatory protein (5). This protein, which has been studied extensively by Stadtman, Rhee, and their colleagues, has no enzymatic activity but rather functions as a protein allosteric effector of the bifunctional enzyme adenylyltransferase/adenylyl-removing enzyme to control the degree of covalent modification and thereby the catalytic activity of glutamine synthetase (6, 7). Thus, PII, which is present in large amounts under conditions of excess nitrogen availability, has two coordinated effects-it causes a decrease in transcription of the ginA gene, which encodes glutamine synthetase, and it causes a decrease in glutamine synthetase catalytic activity (reviewed in ref. 8). Under conditions of limiting nitrogen availability, PI, is covalently modified by a metabolic sensing protein that uridylylates it, and the fully uridylyated form has effects that are essentially opposite to those of the free form (6, 7).Studies of Ronson et al. (9) indicated that the NTRB-NTRC system is likely to be a paradigm for a number of two-component regulatory systems in a wide variety of eubacteria. One component of these systems, for which NTRB is an example...

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.

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