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Abstract
PutA from Escherichia coli functions both as a membrane-associated multifunctional enzyme and a transcriptional repressor of the put regulon, which encodes the putA and putP genes. The intracellular localization and function of PutA is regulated by the availability of proline.
In this study, we investigated how PutA represses the put regulon in the absence of proline. First, we localized the PutA-DNA binding domain to the N-terminal 47 amino acids and clearly demonstrated that PutA belongs to a member of the ribbon-helix-helix (RHH) family transcription factors. Second, we identified five PutA binding sites on the put control DNA region and studied the roles of these binding sites in the regulation of the putA or putP genes. We found that PutA binds to two of the binding sites to repress the putP gene and the other three sites to repress the putA gene expression.
We also explored how PutA switches its localization and function in the presence of proline. Proline reduces the PutA flavin cofactor which induces PutA-membrane binding, where PutA catalyzes the two-step oxidation of proline to glutamate. The PutA-membrane interaction probably leads to a conformational change so that PutA can't interact with DNA, therefore, releasing the repression of the put genes.
Further, we studied how the put genes are regulated by carbon and nitrogen sources. We provided the first evidence that the cAMP receptor protein (CRP) regulates the put genes as an activator by directly interacting with the put control DNA. We also explored whether the nitrogen assimilation control protein (NAC) regulates the put genes.
