| Description |
This dissertation covers two separate research projects that are intellectually connected in that they both deal with studies on the heat shock response in Escherichia coli. The main project of this dissertation has focused on structure/function analysis of the DnaJ heat shock protein to help elucidate the molecular mechanisms by which the DnaJ and DnaK molecular chaperones function together. My results clearly show that the J-domain and G/F motif at the amino terminus of DnaJ interact with DnaK and regulate its function. Specifically, the J-domain stimulates the hydrolysis of ATP bound to DnaK and regulates DnaK's conformational state in an ATP-dependent fashion. In turn, the G/F motif is necessary to orchestrate effective substrate binding to DnaK, mediated by ATP hydrolysis. Hence both of these domains/modules of DnaJ are essential for DnaK's substrate binding activity in the presence of ATP. Thus, for at least some substrates, for example sigma32, DnaJ can catalytically activate" DnaK to form a stable complex, DnaK-sigma32. In addition, I have shown that this regulatory role of DnaJ is necessary and sufficient to elicit DnaJ chaperone function for lambda DNA replication and to negatively regulate the heat shock response. The second project involved the characterization of an insertion mutation in the E. coli cydAB operon, which encodes cytochrome d, was isolated in a screen for new heat shock genes. Null alleles of the cydAB operon were found to exhibit very slow growth at elevated temperature, consistent with the finding that cydAB mRNA accumulates following a temperature upshift. Thus, cydAB is a heat shock operon. Interestingly, the cydAB operon was found to be under the novel heat shock regulation of the ArcA/ArcB two-component regulatory system. In related work, arcA and arcB null mutations were found as extragenic suppressers of cyd mutations. Most likely, such extragenic suppression of the Cyd defect is exerted through the derepression of cyo operon expression, which encodes an alternative cytochrome, o. Consistent with this interpretation, the cyo operon when cloned on a multicopy plasmid can suppress the various Cyd mutant phenotypes. |
