MECHANISM OF TRP GENE REGULATION BY TRAP-RNA RECOGNITION

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Numerous studies have provided a detailed understanding of how regulatory proteins interact with DNA, however relatively little is known about the principles that dictate how sequence-specific RNA-binding proteins recognize their RNA targets. Since fragile X syndrome is caused by defects in an RNA-binding protein, and RNA-binding proteins are involved in regulating HIV and adenovirus gene expression, results from the proposed studies could have a Positive impact on human health. TRAP of Bacillus subtilis is responsible for regulating expression of the trpEDCFBA operon and trpG by binding to a series of closely spaced G/UAG repeats present in each transcript. TRAP binds to a segment of the trp leader RNA that includes the antiterminator, thereby promoting transcription termination. TRAP also binds to a segment of the trpG transcript that includes the trpG ribosome binding site (RBS). The mechanisms responsible for these regulatory events will be characterized using a combination of genetic and biochemical approaches. The first aim of the proposed research is to determine the nucleotides in the G/UAG repeat sequences that are required for TRAP binding, to define the nucleotide spacing requirements between adjacent repeats, and to determine the number of repeats that are necessary to form a stable TRAP-RNA complex. This will be accomplished by testing the ability of various artificial RNA targets to serve as TRAP binding sites in a filter binding assay. Similar experiments will be performed to determine if TRAP can bind to double-stranded RNA in addition to single-stranded RNA. The next aim is to demonstrate that TRAP binding to the trpG RBS prevents translation. RNA footprint analyses will be performed to identify the nucleotides of the trpG transcript that TRAP and ribosomes interact with, and to demonstrate that bound TRAP prevents ribosome binding. In vitro translation studies will be carried out to determine if TRAP binding inhibits TrpG synthesis. The next goal is to determine the time frame in which TRAP binds to the nascent trp leader transcript. If TRAP binding occurs after the antiterminator is formed it is likely that RNA polymerase pauses following its formation to prevent readthrough past the termination signal. In the absence of pausing, TRAP binding must be sufficiently rapid to prevent antiterminator formation. Single-round transcription experiments will be carried out in vitro to determine if RNA polymerase pauses following antiterminator formation. NusA protein will be added to the transcription system to see if it plays a role in polymerase pausing. Evidence for pausing will also be examined in vivo. Lastly, a structure-function analysis will be performed to identify amino acid residues of TRAP that are responsible for RNA binding, L-tryptophan binding, and subunit oligomerization, as well as to identify nucleotides that interact with specific amino acid residues of TRAP. TRAP deficient mutants will be obtained using several strategies, including genetic selections and site-directed methods. The defects associated with various mutant TRAP proteins will be determined using available techniques. A collaboration with William Royer will continue to determine the crystal structure of the TRAP-RNA complex.