Genetic analysis of the GcvA binding site in the gcvA control region

The GcvA protein both activates and represses the gcv operon and negatively regulates its own transcription. GcvA binds to three sites in the gcv cont rol region and to one site in the gcvA control region; each of these bindin g sites contains the conserved 5 bp DNA sequence 5'-CTAAT-3'. This report d escribes the role this DNA sequence plays in autoregulation and expression of gcvA. Through single base-pair mutations, the importance of three of the se five basepairs in the autoregulation of gcvA expression is shown. Two of the gcvA control region mutations described cause a gcvA::lacZ fusion to b e overexpressed at 9-24 times the wild-type level. The increase in expressi on is due in part to a complete loss of autoregulation and in part to a Gcv A-independent mechanism. One of the mutants was shown by Western blot analy sis to increase the intracellular concentration of GcvA. This high level of gcvA expression subsequently causes the loss of purine-mediated repression of a gcvT::lacZ fusion. However, overexpression of gcvR re-established pur ine-mediated repression of the gcvT::lacZ fusion, supporting the model for gcv regulation that suggests the need for a relatively constant GcvA to Gcv R ratio for appropriate regulation of gcv expression in response to glycine and purine availability.