The long term goal of this work is to elucidate the means of regulation of the SpoT enzyme(s) of Escherichia coli. The spoT gene encodes an enzyme or enzymes possessing guanosine tetraphosphate (ppGpp) synthesis activity (PSII enzyme) and ppGpp degradation activity (hydrolase enzyme). Previous work has suggested three things. (1) the PSII enzyme is unstable, (2) uncharged tRNA inhibits the hydrolase, and (3) a regulatory domain may reside in the C-terminal half of the hydrolase. This project seeks to resolve whether each of these is true. For the PSII stability question, antisense RNA will be used to specifically shut down the translation of the spoT mRNA. They hydrolase activity is stable, so if translation of spoT is stopped, and PSII is unstable, then ppGpp levels will decline since ppGpp synthesis stops while ppGpp degradation continues. If PSII is unstable, as expected, then investigations into the regulation of spoT translation will be done. In addition, the relative contributions of PSI (relA gene product) and PSII to ppGpp levels during exponential growth will be determined. Items (2) and (3) above are related, and probably only one of these will be resolved by this work. Recent work has shown that a truncated spoT gene produces hydrolase activity, but not PSII activity (PS pi alleles). It is not known whether the hydrolase activity produced from this allele is regulated like wild type or not. If the hydrolase is regulated normally in this mutant, then such a mutant provides an excellent means of testing whether uncharged tRNA inhibits the hydrolase in vivo. If this is shown to be so, then experiments will also be done to check for an in vitro binding of uncharged tRNA to SpoT. If the hydrolase is not regulated normally in the PS phi mutant, then the truncated region has defined an area of the protein which is important for regulation, and attempts will be made to more precisely define the regulatory site.
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