The Escherichia coli glycine cleavage enzyme system catalyzes the cleavage of glycine, generating CO 2 , NH 3 , and a one-carbon unit. Expression of the operon encoding this enzyme system (gcv) is induced in the presence of glycine and repressed in the presence of purines. In this study, a mutant with high-level constitutive expression of a gcvT-lacZ gene fusion was isolated. The mutation in this strain was designated gcvR1 and was mapped to min 53.3 on the E. coli chromosome. A single-copy plasmid carrying the wild-type gcvR gene complemented the mutation, restoring normal regulation of a gcvT-lacZ fusion, while a multicopy plasmid carrying gcvR led to superrepression under all growth conditions. Negative regulation of a gcvT-lacZ fusion by GcvR was shown to require GcvA, a LysR family protein known to both activate gcv in the presence of glycine and repress gcv in the presence of purines. Models explaining how GcvR and GcvA might interact to regulate gcv expression are proposed.The glycine cleavage enzyme system catalyzes the oxidative cleavage of glycine into CO 2 and NH 3 and transfers the onecarbon (C 1 ) methylene unit to tetrahydrofolate, forming 5,10-methylenetetrahydrofolate (8). This C 1 -containing molecule can then be used as a C 1 donor in the biosynthesis of purines, methionine, thymine, and numerous methylated products (14). In balancing cellular requirements for glycine and C 1 units, the glycine cleavage enzyme system serves an important role in cellular metabolism.The Escherichia coli glycine cleavage enzyme system consists of four proteins. The T, H, and P proteins are encoded by the gcv operon, which maps at min 62.5 on the E. coli chromosome (15, 21). The L protein, encoded by the lpd gene, is not in the gcv operon; the lpd gene maps at min 2.5 (25).So far, three regulatory proteins, Lrp, PurR, and GcvA, have been shown to be involved in regulation of gcv expression. Lrp, or leucine-responsive regulatory protein, is a global regulatory protein involved in the control of transcription of numerous genes involved in amino acid metabolism (3). It was shown that in E. coli cells carrying a fusion in which ␤-galactosidase synthesis is under the control of the gcv regulatory region, a mutation in lrp results in a low level of noninducible ␤-galactosidase synthesis (10, 23).The PurR protein is a negative regulator of many genes involved in nucleotide metabolism, including gcv (6,9,16,24,28). PurR mediates a twofold reduction both in Gcv enzyme levels (21) and in ␤-galactosidase synthesis in cells carrying a gcvT-lacZ fusion in response to purine supplementation (28). PurR-mediated repression of gcv occurs at the transcriptional level (21), and gel mobility shift assays and DNase I footprint assays have indicated that the purified PurR protein binds to the gcv control region, overlapping the gcvT transcription start site (28).The GcvA protein activates expression of gcv in vivo in glycine-supplemented cultures (29) and is also necessary for a 10-fold, PurR-independent repression of gcv when cells are...

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Plasmid pGS146 carries the Escherichia coli gcv system on a 7.12 kb SalI-BamHI DNA insert fragment. The DNA sequence of a gene which presumably encodes the T-protein of the glycine cleavage (GCV) enzyme complex was determined. The gene, designated gcvT, encodes a polypeptide of 364 amino acids with a calculated molecular weight of 40,146 daltons. In a minicell system, the SalI-BamHI fragment directs the synthesis of three polypeptides with Mr values of about 93,300, 43,300 and 17,400 daltons. When gcvT was inactivated by insertion of a translation terminator sequence, the Mr 43,300 dalton polypeptide was not observed. The deduced amino acid sequence of the E. coli T-protein was compared with the sequence of the T-protein from bovine liver. 190 of 364 amino acid residues are identical or chemically similar between the two proteins. An S1 nuclease mapping experiment located the transcription start point for gcvT. Single basepair changes were made in the promoter -10 and -35 sequences. These mutations significantly reduced expression from a gcvT-lacZ gene fusion. The gcvT gene is transcribed and translated in the same direction as the gcvH gene.

The Escherichia coli glycine cleavage enzyme system, encoded by the gcvTHP operon, catalyses the oxidative cleavage of glycine to CO 2 , NH 3 and a onecarbon methylene group. Transcription of the gcv operon is positively regulated by GcvA and negatively regulated by GcvA and GcvR. Using a LexAbased system for analysing protein heterodimerization, it is shown that GcvR interacts directly with GcvA in vivo to repress gcvTHP expression. Several mutations in either gcvA or gcvR that result in a loss of gcv repression also result in a loss of GcvA/GcvR heterodimerization. Finally, it is shown that the C-terminal half of GcvA is involved in its interaction with GcvR, whilst the entire GcvR protein appears to be necessary for heterodimerization.

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