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...