The GroE heat shock proteins (GroEL and GroES) of <i>Escherichia coli</i> represent major molecular chaperones that participate in folding and assembly of a variety of proteins and are essential for cell growth at all temperatures. From <i>in vitro</i> studies, GroEL is thought to be highly promiscuous in substrate binding, interacting with almost any non-native model protein. However, <i>in vivo</i>, GroEL is involved in the folding of only 10-15% of newly translated polypeptides, suggesting specificity for a defined set of substrates. In an attempt to identify GroE substrates, a strain of <i>E. coli</i> has been constructed in our laboratory in which expression of GroE can be turned off. Previous work from our laboratory had suggested that DapA, the first enzyme on the lysine biosynthetic pathway, was affected by GroE depletion in <i>Escherichia coli</i>. Native DapA is present as a homotetramer; in this study, work is described that suggests that DapA achieves a homotetrameric state in GroE depleted cells but lacks enzymatic activity. Work is also described that suggests that native DapA cannot be overproduced in GroE depleted cells as it becomes highly susceptible to aggregation. Work in chapter 4 suggests that the <i>lac</i> inhibitor protein, LacI, is also affected by GroE depletion. <i>In vitro</i> footprinting studies in GroE depleted cells suggest that LacI remains bound to the <i>lac</i> operator sequence in the presence of the gratuitous inducer IPTG. Previous work in other laboratories had suggested that <i>dapA </i>is not regulated at the level of expression. In this work data is presented that suggests that <i>dapA </i>is regulated at the level of expression, possibly by a transcriptional activator, in response to intracellular levels of the molecule diaminopimelic acid.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:640148 |
| Date | January 2001 |
| Creators | Acord, John |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/10743 |
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