No abstract

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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We previously isolated a mutant of Escherichia coli that is preferentially affected in the synthesis of rRNA and has a mutation in the gene (accD) encoding a subunit of acetyl-CoA carboxylase. Using this mutant and other mutants of the pathway for fatty acid and phospholipid biosynthesis as well as cerulenin, a specific inhibitor of fatty acid synthesis, we show that (i) inhibition of fatty acid synthesis in the presence of both a carbon source and all 20 amino acids stimulates the accumulation of guanosine tetraphosphate (ppGpp) and leads to preferential inhibition ofrRNA synthesis, (ii) this ppGpp accumulation is spoT dependent, and (iii) the generation of the metabolic signal that stimulates this spoTmediated response probably does not depend on either phospholipid starvation or a significant reduction in the level of ATP.It is well known that amino acid starvation in Escherichia coli leads to accumulation of guanosine tetraphosphate (ppGpp) and cessation of rRNA synthesis. This (about 0.2-0.3 mM) medium. Samples (0.1 ml) were mixed with 10 ul of 11 M formic acid on ice, and cell debris and inorganic phosphate were removed as described (11). Equal volumes of these formic acid extracts were chromatographed in one or two dimensions on polyethyleneimine (PEI) cellulose TLC plates (11, 12). For quantification ofppGpp, an area equal in size to each ppGpp spot between ppGpp and GTP Abbreviations: ACP, acyl-carrier protein; ts, temperature sensitive.

Many of the changes in gene expression observed when Escherichia coli cells enter stationary phase are regulated at the level of transcription initiation. A group of stationary-phase-inducible promoters, known as "gearbox" promoter, display a characteristic sequence in the -10 region which differs greatly from the consensus sequence for sigma 70-dependent promoters. Here we describe our studies on the gearbox promoters bolAp1 and mcbAp, responsible for the temporally regulated transcription of bolA and the genes involved in the synthesis of the peptide antibiotic microcin B17, respectively. Deletion analysis of mcbAp demonstrated that the stationary-phase-inducible properties of this promoter are found in a DNA fragment extending from -54 to +11 bp, surrounding the transcriptional start site, and are separable from DNA sequences responsible for the OmpR-dependent stimulation of transcription of mcbAp. In vitro transcription studies indicate that the RNA polymerase holoenzyme involved in the transcription of mcbAp contains sigma 70. In this and an accompanying paper (R. Lange and R. Hengge-Aronis, J. Bacteriol. 173: 4474-4481, 1991), experiments are described which show that the product of katF, a global regulator of stationary-phase gene expression and a putative sigma factor, is required for the expression of bolAp1 fused to the reporter gene lacZ. In contrast, mcbAp appears to be negatively regulated by katF. We discuss the implications of these results for postexponential gene expression and the role of gearbox sequences in the regulation of promoter activity.

Five purified protein components, RNA polymerase I, Rrn3p, core factor, TBP (TATA-binding protein), and upstream activation factor, are sufficient for high level transcription in vitro from the Saccharomyces cerevisiae rDNA promoter. Rrn3p and pol I form a complex in solution that is active in specific initiation. Three protein components, pol I, Rrn3p, and core factor, and promoter sequence to ؊38, suffice for basal transcription. Unlike pol II and pol III, yeast pol I basal transcription does not require TBP. Instead, TBP, upstream activation factor, and the upstream element of the promoter together stimulate pol I basal transcription to a fully activated level. The role of TBP in pol I transcription is fundamentally different from its role in pol II or pol III transcription.Of the three nuclear RNA polymerases, it is RNA polymerase I (pol I) 1 that synthesizes large rRNAs. In Saccharomyces cerevisiae, a precursor 35 S rRNA is transcribed and then processed into the mature 18 S, 5.8 S, and 25 S rRNAs found in ribosomes. These rRNAs are encoded by 100 -200 direct rDNA repeats on chromosome XII. Each spacer region between the pol I-driven 35 S transcription units contains a gene encoding the remaining rRNA, 5 S rRNA, transcribed by pol III.The only essential function of pol I in yeast is synthesis of the 35 S rRNA transcript, since the lethal phenotype of a deletion in the second largest subunit of pol I can be rescued by synthesis of the 35 S rRNA transcript by pol II from a GAL promoter placed correctly upstream of the 35 S transcription unit on a high copy plasmid (1). This provided a screen for mutants dependent on pol II-driven synthesis of rRNA from the GAL promoter (2). Such rrn mutants were expected to be defective in pol I activity in vivo, and confirming this, mutations in genes encoding subunits of pol I (those not shared with either pol II or pol III) were isolated (2). Other mutations that also caused defects in rRNA synthesis (as assessed by pulse labeling in vivo) eventually proved to lie in genes encoding (subunits of) pol I transcription factors.An important advance in the study of yeast pol I was the development of an in vitro transcription system using a crude extract (3-5) and, later, fractionated extracts (6 -8). Extracts from rrn mutant strains were not active, and their activity could be restored by addition of fractions from a wild-type extract. This was used as an assay for the purification of pol I transcription factors (6). The availability of cloned RRN genes, which could be tagged with the hemagglutinin antigen (HA) or hexahistidine, greatly facilitated purification. In this way, the multi-subunit factors, core factor (CF), and upstream activation factor (UAF), and the single subunit factor Rrn3p were identified and shown to be necessary for activity in the crude in vitro system as well as in vivo (6, 9 -12).Like higher eukaryotes, the yeast pol I promoter is composed of a core element that is essential for transcription, located roughly between ϩ5 and Ϫ40 relative to the start site ...