Recently, a unique regulatory element in the transcription of tRNA synthetases in Gram-positive bacteria was identified. A novel RNA-RNA interaction occurs between uncharged tRNA and the mRNA 5' leader region of many Gram-positive tRNA synthetases. This interaction leads to antitermination of transcription and complete read-through of the gene. Without this interaction (i.e. in the presence of only charged tRNA), transcription termination occurs. The sequence and secondary structure dependence of this antitermination indicates a definite, sequence dependent interaction. However, based on these studies, it also appears as though the overall three-dimensional structure of the leader region and its complex with the uncharged tRNA plays a critical role in the antitermination function. The hypothesis is that there is a crucial tertiary structure/function correlation in the antiterminator bulge portion of the leader region. By studying structures of mutant sequences with decreased antitermination ability compared to the wild type, Dr.Hines can begin to construct a structure/function relationship. With further structural information, she can look at changes upon interaction with tRNA and begin to assay for and propose drug inhibitors. The long-range goal of this project is to disrupt the tRNA/mRNA interaction and function with small molecules that have been targeted to this system, using the structural information obtained in these studies. Such studies will lead to the development of novel antibiotics.
Specific Aim I : Dr. Hines will investigate the solution structure of antiterminator bulge mutants where the mutation has been implicated by genetic studies to play a functional role. The structure of the mutants will be compared to the wild-type bulge in order to add to the knowledge of structure/function relationships for this system.
Specific Aim II : Using either fully modified tRNA or a simplified tRNA acceptor stem model RNA she will investigate the solution behavior of the tRNA/antiterminator bulge interaction. She will investigate this interaction using native gels, fluorescence and NMR.
Specific Aim III : She will determine tRNAIbulge sequence and structural requirements for functional interactions in vitro and antitennination in vivo.
Specific Aim I V: She will begin to look at the effects small RNA binding ligands may have on the solution behavior of the antiterminator alone or complexed with tRNA acceptor stem.
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