Regulation of gene expression by repressor localization: biochemical evidence that membrane and DNA binding by the PutA protein are mutually exclusive
Journal of BacteriologyPublished 1 April 1997Open access
Alicia M. Muro‐Pastor, Paula Ostrovsky, Stanley Maloy
Citations70
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TL;DR
The results indicate that wild-type PutA does not simultaneously associate with DNA and membranes, and support a model in which the PutA protein shuttles between the membrane and the cytoplasm, depending on the availability of proline.
Abstract
The PutA protein from Salmonella typhimurium is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate, a reaction that is coupled to the transfer of electrons to the electron transport chain in the cytoplasmic membrane. The PutA protein is also a transcriptional repressor that regulates the expression of the put operon in response to the availability of proline. Despite extensive genetic and biochemical studies of the PutA protein, it was not known if the PutA protein carries out both of these two opposing functions while membrane associated or if instead it carries them out in different cellular compartments. To distinguish between these alternatives, we directly assayed the binding of purified PutA protein to DNA and membranes in vitro. The results indicate that wild-type PutA does not simultaneously associate with DNA and membranes. In addition, PutA superrepressor mutants that exhibit increased repression of the put genes show a direct correlation between decreased membrane binding and increased DNA binding. These results support a model in which the PutA protein shuttles between the membrane (where it acts as an enzyme but lacks access to DNA-binding sites) and the cytoplasm (where it binds DNA and acts as a transcriptional repressor), depending on the availability of proline.
Keywords
Materials ScienceNursingBiochemistry, Genetics and Molecular Biology
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