NOVEL CONTROL MECHANISMS IN ENDOSPORE FORMATION

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This proposal is for research into the molecular details of regulation of the Bacillus subtilis pyrimidine nucleotide (pyr) biosynthetic operon by a novel autogenous transcription attenuation mechanism. The first gene of the 10 cistron pyr operon encodes a protein, PyrR, that acts to promote termination of transcription at three separate attenuation sites in the operon when exogenous pyrimidines are present in the growth medium. Experimental results obtained so far support a model for attenuation in which PyrR acts by binding in a UMP-dependent manner to specific sequences in pyr mRNA and favoring formation of a transcription terminator structure by preventing formation of a competing antiterminator structure. Experiments are proposed to test rigorously and to refine this model both in vitro and in vivo. An extraordinary feature of the PyrR regulatory protein is that it is also a uracil phosphoribosyltransferase with little sequence similarity to other uracil phosphoribosyltransferases. The PyrR protein, which has been purified to homogeneity and crystallized, will be fully characterized; its structure will be determined at high resolution. The relationship between the enzymatic activity of PyrR and its ability to regulate pyr transcription will be analyzed by biochemical and mutational studies. The primary and secondary structure of RNA sequences specifically bound by the pure PyrR protein will be studied. If cocrystallization can be achieved, the structure of a PyrR-RNA complex at high resolution will be determined. The regulation of pyr transcription in vitro by PyrR will be investigated using purified components. The secondary structures of pyr mRNA attenuators in the absence and presence of PyrR will be characterized. The regulation of attenuation at each of the three termination sites by pyrimidines and during sporulation will be investigated in vivo by overproduction of specific RNA fragments. Mutants in PyrR and in the attenuator sequences are being obtained by in vivo selection; these and mutants prepared by in vitro mutagenesis will be characterized. This research is focused on the fundamental nature of a novel and potentially very general regulatory mechanism, but it may also have eventual application of the development to new types of anti- microbial agents or to improvements in biotechnological production.