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

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Abstract
The GcvA protein both activates and represses the gcv operon and negatively regulates its own transcription. GcvA binds to three sites in the gcv control region and to one site in the gcvA control region; each of these binding sites contains the conserved 5 bp DNA sequence 5'-CTAAT-3'. This report describes the role this DNA sequence plays in autoregulation and expression of gcvA. Through single base-pair mutations, the importance of three of these five basepairs in the autoregulation of gcvA expression is shown. Two of the gcvA control region mutations described cause a gcvA:: lacZ fusion to be overexpressed at 9-24 times the wild-type level. The increase in expression is due in part to a complete loss of autoregulation and in part to a GcvA- independent mechanism. One of the mutants was shown by Western blot analysis to increase the intracellular concentration of GcvA. This high level of gcvA expression subsequently causes the loss of purine-mediated repression of a gcvT: : lac2 fusion. However, overexpression of gcvR re-established purine- mediated repression of the gcvT::lacZ fusion, supporting the model for gcv regulation that suggests the need for a relatively constant GcvA to GcvR ratio for appropriate regulation of gcv expression in response to glycine and purine availability.

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