We study regulation of gene expression in a variety of microbes, particularly under conditions of environmental stress. Our work is highly interdisciplinary, invoking genetics, biochemistry, structural biology and chemical engineering.
First, we study the biology of introns, dynamic sequences that interrupt genes and can therefore disrupt the flow of genetic information. The work runs the gamut from answering fundamental questions about how introns function and how they are removed to preserve genetic integrity, through how introns might have evolved, to ways in which they could be exploited in biotechnology. Introns exist in almost all life forms, from simple bacteria to more complex species, including humans. Our studies are based on the discovery at Wadsworth Center in 1984 that, contrary to then current dogma, introns exist in simple organisms. The conservation of introns across species allows their study in model organisms, with the attendant advantages of rapid and refined experimentation. We investigate several types of introns with two properties in common. First, they are removed at the level of the RNA by a process called splicing. The intron RNAs are self-splicing; they are themselves the enzymes, called ribozymes, that catalyze splicing. Second, the introns also can move at the level of the DNA, acting as mobile genetic elements. Both the RNA splicing and the DNA mobility mechanisms are examined, as are the proteins that assist these reactions. These studies are based on genetic and biochemical analyses, as well as collaborative structural approaches involving X-ray crystallography and NMR. We have elucidated the different molecular pathways whereby introns splice and move by recruiting proteins of unusual structure and function. The two intron types under study, group I and group II, splice and move using different mechanisms. We have shown that whereas group I introns move or “home” by a DNA-based double-strand break repair mechanism, group II introns move via an RNA intermediate in processes termed “retrohoming” (to homologous sites) and "retrotransposition" (to ectopic sites). Thus, the group II introns resemble retrotransposons, a finding that raises the possibility of their use as delivery vehicles for gene therapy. Our investigations have also posed evolutionary questions and shed light on the origin and persistence of mobile introns.
Second, unraveling the structure and function of inteins, a type of intervening sequence that is remarkable for splicing at the protein level, is another focus of the Belfort laboratory. Their practical applications are being explored, too, and we hold patents for the use of both introns and inteins in biotechnology. Both elements can be used to facilitate protein purification, while inteins, which are found in critical genes of human microbial pathogens, are promising targets for development of novel antibiotics. Developing intein-base anti-tuberculosis drugs is one of the current interests of the laboratory.
Third, we have a longstanding interest in small RNAs (sRNAs). After performing fundamental studies on sRNA regulation in E. coli, we have recently turned our attention to sRNAs in Mycobacteria and their potential role in regulating pathogenicity.