The Escherichia coli gcvB gene encodes a small RNA transcript that is not translated in vivo. Transcription from the gcvB promoter is activated by the GcvA protein and repressed by the GcvR protein, the transcriptional regulators of the gcvTHP operon encoding the enzymes of the glycine cleavage system. A strain carrying a chromosomal deletion of gcvB exhibits normal regulation of gcvTHP expression and glycine cleavage enzyme activity. However, this mutant has high constitutive synthesis of OppA and DppA, the periplasmic‐binding protein components of the two major peptide transport systems normally repressed in cells growing in rich medium. The altered regulation of oppA and dppA was also demonstrated using oppA–phoA and dppA–lacZ gene fusions. Although the mechanism(s) involving gcvB in the repression of these two genes is not known, oppA regulation appears to be at the translational level, whereas dppA regulation occurs at the mRNA level.

Nitric oxide (NO) is associated with broad-spectrum antimicrobial activity of particular importance in infections caused by intracellular pathogens. An insertion mutation in the metL gene of Salmonella typhimurium conferred specific hypersusceptibility to S-nitrosothiol NO-donor compounds and attenuated virulence of the organism in mice. The metL gene product catalyzes two proximal metabolic steps required for homocysteine biosynthesis. S-Nitrosothiol resistance was restored by exogenous homocysteine or introduction of the metL gene on a plasmid. Measurement of expression of the homocysteine-sensitive metH gene indicated that S-nitrosothiols may directly deplete intracellular homocysteine. Homocysteine may act as an endogenous NO antagonist in diverse processes including infection, atherosclerosis, and neurologic disease.

In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria–Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA expression in Luria–Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified a region complementary to this region of cycA mRNA. However, mutations predicted to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

ABSTRACr We have isolated two regulatory mutants altered in the leader region of the Escherichia coli tryptophan (tp) operon. In one mutant, trpL29, the AUG translation start codon for the tip leader peptide is replaced by AUA. The other mutant, trpL75, has a G-A change at residue 75, immediately after the UGA translation stop codon for the trp leader peptide. In vivo, trpL29 and trpL75 increase the efficiency of transcr!ption termination at the tip attenuator 3-to 5-fold. trpL29 and tpL75 also fail to respond fully to tryptophan starvation and other conditions that normally relieve transcription termination at the tip attenuator. The tipL29 mutation, which presumably reduces synthesis of the tip leader peptide, is cis dominant. The effect of starvation for a number of the amino acids in the tp leader peptide was determined. Only starvation for tryptophan and arginine, amino acids that occur at residues 10, 11, and 12 of the 14-residue tip leader peptide, elicits relief of transcription termination. Our findings suggest that translation of tip leader RNA is involved in regulation of transcription termination at the attenuator. A mode isiscussed in which the location of the ribosome synthesizing the leader peptide is communicated to the RNA polymerase transcribing the leader region.RNA polymerase molecules that have initiated transcription at the promoter of the tryptophan (trp) operon of Escherichia coil may either terminate transcription at the attenuator, or a site, in the 160-base-pair leader region of the operon or continue transcription into the structural genes (1). Termination of transcription at trp a is regulated, and varies in response to changes in the levels of charged vs. uncharged tRNATrP (2). We define attenuation as the regulation of this termination (3).The short RNA molecules produced by transcription termination at an attenuator are termed leader transcripts. The known leader transcripts of amino acid biosynthetic operons code for short peptides containing at least two tandem amino acid residues that are the end product of expression of that operon (refs. 4-7; unpublished results

The GcvA protein is a LysR family regulatory protein necessary for both activation and repression of the Escherichia coli glycine cleavage enzyme operon (gcv) and negative regulation of gcvA. Gel shift assays indicated that overexpressed GcvA in crude extracts is capable of binding specifically to DNA containing the gcv and gcvA control regions. DNase I footprint analysis of the gcvA control region revealed one region of GcvA-mediated protection overlapping the transcription initiation site and extending from ؊28 to ؉20. Three separate GcvA binding sites in gcv were identified by DNase I footprint analysis: a 29-bp region extending from positions ؊271 to ؊242, a 28-bp region extending from ؊242 to ؊214, and a 35-bp region covering positions ؊69 to ؊34 relative to the transcription initiation site. PCR-generated mutations in any of the three GcvA binding sites in gcv decreased GcvA-mediated activation and repression of gcv.The Escherichia coli glycine cleavage enzyme system (GCV) is a glycine-inducible, purine-repressible metabolic pathway that catalyzes the cleavage of glycine into CO 2 ϩ NH 3 and transfers a one-carbon (C 1 ) methylene unit to tetrahydrofolate (9,15). This activated methyl group, in the form of 5,10-methylenetetrahydrofolate, can then be used in the biosynthesis of purines, methionine, thymine, and other cellular components (17). Three of the four enzymes required for glycine cleavage, the T, H, and P proteins, are encoded by the gcv operon that maps at min 65.2 on the E. coli chromosome (20). Expression of the gcv operon involves a complex regulatory system consisting of at least three regulatory proteins acting at the gcv promoter.Two regulators known to play a negative role in the control of gcv expression are the PurR and GcvA proteins (36). PurR is required for negative regulation of numerous genes involved in nucleotide metabolism (7,10,21,34) and has been shown to decrease gcv expression twofold when cells are grown in the presence of exogenous purines or purines and glycine (36). In vitro studies have indicated that PurR binds to the gcv control region near the transcription initiation site for gcvT, the first gene in the gcv operon, and presumably interferes with the ability of RNA polymerase to initiate transcription (28,36). A PurR-independent mechanism for purine repression of gcv is mediated by the GcvA protein. This LysR-type regulator (8, 25) is necessary for an additional fivefold decrease in gcv expression when cells are grown in the presence of purines and without glycine (36).GcvA plays a dual role in the regulation of gcv, since it is also required for six-to sevenfold activation of gcv when glycine is included in the growth media (37). Whether GcvA responds directly to two different cellular metabolites or whether GcvA interacts with another protein is unknown but is under investigation. GcvA also negatively regulates its own promoter independently of glycine or purine supplementation.Full induction of gcv in response to glycine also requires Lrp (12), a global regulator ne...