We have continued studies on the role that energy-dependent protein degradation plays in regulating gene expression, using Escherichia colias a model system. RssB, a protein that regulates the degradation of the stationary phase sigma factor RpoS has been found to present RpoS to the ClpXP protease. Degradation is signalled by phosphorylation of RssB; we are investigating the mechanism of phosphorylation and dephosphorylation. In addition to regulation of RpoS degradation, RpoS translation is regulated by DsrA, a small stable 85 nucleotide RNA. A stem-loop at the 5 end of the RNA is necessary for regulation of RpoS, and acts by pairing with the RpoS leader. This disrupts the secondary structure of the RpoS leader, allowing translation. The promoter of dsrAis regulated by temperature (on at low temperatures, off at high temperatures). We find that temperature regulation resides in a minimal promoter region of 36 base pairs, suggesting that promoter structures may be mediating temperature regulation. A novel new RNA, RprA, has been identified as a suppressor of dsrAmutants. It also acts to stimulate RpoS translation, although the mechanism of action appears to differ from that for DsrA. Both DsrA and RprA participate in the osmotic shock induction of RpoS. Osmotic shock appears to modulate their action rather than their synthesis. The demonstration of a second small RNA regulator of RpoS, in addition to DsrA, supports the idea that multiple small RNAs may mediate translational regulation in many cases. In mutagenesis studies of the Lon protease, we find that deletions and point mutations in the C- terminal domain that remove or inactivate the proteolytic active site are still able to partially complement lon mutants when overproduced. This appears to be because the substrate binding domain is retained in the N-terminus of the deleted proteins, and binding of substrate is sufficient to interfere with substrate function, mimicking the effect of degradation. However, binding to the deleted proteins protects substrates from secondary proteases. This will allow the analysis of substrate recognition and binding by the Lon protease to be studied independently of protein degradation. Further analysis of Lon domains was carried out by partial proteolysis and by using regions of protease sensitivity as the boundaries for separately expressed Lon domains. By analysis of the in vivo and in vitro properties of these fragments, we are beginning to understand the organization of this energy-dependent protease. Similar approaches are being used to analyze the ClpA ATPase of the ClpAP protease. - Escherichia coli, Gene regulation, heat shock proteins, prokaryotes, proteases, transcriptional control, regulatory RNA, - Neither Human Subjects nor Human Tissues
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