The specific and accurate attachment of amino acids onto tRNA molecules is a critical step in protein biosynthesis. RNA recognition and discrimination by aminoacyl-tRNA synthetases play essential roles in this process. This research is aimed at understanding how these important enzymes recognize and interact with their tRNA substrates. Computer-aided sequence comparisons of the primary structures of synthetases from a variety of organisms have resulted in the division of the twenty enzymes into two classes. The ten enzymes designated as """"""""class II"""""""" have been further divided into sub-classes IIa and IIb. Escherichia coli proline tRNA synthetase (ProRS) is the representative protein in class IIa, being most closely related to the other members. The objective of this research is to investigate how this important class II enzyme functions in specific recognition.
The Specific Aims of this work are: (1) Identification of RNA structural requirements and specific RNA functional groups important in aminoacylation by E. coli ProRS. Footprinting techniques and mutant tRNAs will be used to continue to elucidate the set of nucleotides that mark a tRNA molecule for specific aminoacylation with proline. Chemical modification of tRNA mutants with reagents such as dimethylsulfate and diethyl-pyrocarbonate will be used to understand the structural basis for some of the observed effects. Semi-synthetic tRNAs will be used to facilitate the identification of RNA structural characteristics as well as specific functional groups that are important for aminoacylation by ProRS; (2) Elucidation of protein structural motifs important for tRNAPro recognition. Site-specific chemical modification of specifically placed cysteine residues will be used to probe class II consensus motifs 2 and 3. Novel applications of chemical RNA synthesis will enable the covalent attachment of probes at specific, internal positions of semi-synthetic tRNA substrates for use in cross-linking and fluorescence studies; (3) Understanding species-specific aminoacylation of tRNAPro. Human and E. coli ProRS share only approximately 20 percent sequence homology. Their experiments will determine if the positional locations of identity nucleotides that specify aminoacylation with proline have been conserved from bacteria to mammals.
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