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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM055875-01
Application #
2024424
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1997-04-01
Project End
2001-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Boston Biomedical Research Institute
Department
Type
DUNS #
058893371
City
Watertown
State
MA
Country
United States
Zip Code
02472
Gangopadhyay, Jaya Pal; Jiang, Shu-Qin; Paulus, Henry (2003) An in vitro screening system for protein splicing inhibitors based on green fluorescent protein as an indicator. Anal Chem 75:2456-62
Gangopadhyay, Jaya Pal; Jiang, Shu-qin; van Berkel, Patrick et al. (2003) In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions. Biochim Biophys Acta 1619:193-200
Lew, Belinda M; Paulus, Henry (2002) An in vivo screening system against protein splicing useful for the isolation of non-splicing mutants or inhibitors of the RecA intein of Mycobacterium tuberculosis. Gene 282:169-77
Paulus, H (2001) Inteins as enzymes. Bioorg Chem 29:119-29
Mills, K V; Paulus, H (2001) Reversible inhibition of protein splicing by zinc ion. J Biol Chem 276:10832-8
Shingledecker, K; Jiang, S q; Paulus, H (2000) Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein. Arch Biochem Biophys 375:138-44
Paulus, H (2000) Protein splicing and related forms of protein autoprocessing. Annu Rev Biochem 69:447-96
Lew, B M; Mills, K V; Paulus, H (1999) Characteristics of protein splicing in trans mediated by a semisynthetic split intein. Biopolymers 51:355-62
Mills, K V; Lew, B M; Jiang, S et al. (1998) Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein. Proc Natl Acad Sci U S A 95:3543-8
Lew, B M; Mills, K V; Paulus, H (1998) Protein splicing in vitro with a semisynthetic two-component minimal intein. J Biol Chem 273:15887-90

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