The objective of this proposal is to elucidate the mechanism of protein splicing in Mycobacterium tuberculosis. Protein splicing, discovered only six years ago, is an extraordinary process by which the flow of information from a gene to its protein product is modulated so as to yield two unrelated proteins. It involves the precise, self-catalyzed excision of an intervening polypeptide sequence from a precursor protein with the concomitant joining of the flanking sequences to produce two new proteins. All information and catalytic groups required for this process reside in the intervening sequence, called the intein. In this proposal, the intein that interrupts the RecA protein of M. tuberculosis will be cloned into a foreign context that allows efficient expression of a self-splicing protein in Escherichia coli. This chimeric construct will be further modified by splitting the intein into two complementary segments that can be recombined in vitro to undergo splicing. Using as guide the ability to undergo splicing and the integrity of the active center as revealed by fluorescent- probes, the catalytic amino acid residues in the intein will be identified by site-directed mutagenesis, whereas elements not essential for the splicing process will be deleted. The interactions of the intein domains essential for protein splicing will be mapped by photo crosslinking, protein footprinting, and peptide competition studies. The results obtained will serve to define the active center of self-splicing inteins, both in terms of the catalytic residues involved and their spatial arrangement. They will thus provide important insights into the biochemical mechanism underlying protein splicing, a novel biological process. Moreover, protein splicing is an obligatory step in the activation of the RecA protein of M. tuberculosis and these results may therefore suggest new strategies for the chemotherapy of one of the most wide-spread infectious diseases. This is because the RecA protein plays an important role in the repair of the damage caused by the reactive oxygen species with which macrophages attempt to kill invading bacteria. Agents that interfere with the splicing of the M. tuberculosis RecA protein may thus constitute a new class of antimycobacterial drugs.
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