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. Recently, 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. Proline tRNA synthetase (ProRS) is the representative protein in class IIa, being most closely related to the other members. To date, however, nothing is known about how this important class II enzyme functions in specific RNA recognition. The primary objective of this research is to address this question. Nucleotides that are critical for the specific recognition of several tRNAs by their respective class II synthetases are clustered within the first four base pairs of the acceptor stem domain. A C1:G72 base pair in the acceptor stem, unique to tRNA(Pro), may be a potential site for synthetase discrimination. Footprinting techniques and mutant full-length tRNAs will be used to identify the set of nucleotides that mark a tRNA molecule for specific aminoacylation with proline. A tRNA molecule can be assembled by annealing together two oligonucleotides representing 3'- and 5'-fragments. For example, it has been shown by the present investigator that a chemically synthesized 5'-18-mer can be combined with an in vitro transcribed 3'-59 mer to form a functional tRNA(Pro) molecule. The use of these """"""""annealed"""""""" substrates, prepared with a chemically-synthesized fragment, will facilitate the identification of RNA structural characteristics that are important for aminoacylation by ProRS. Modified bases and single deoxynucleotide substitutions will enable critical functional groups to be identified. The objective of this work is to develop a detailed, molecular-level understanding of how ProRS recognizes and interacts with its tRNA substrate. Novel applications of chemical RNA synthesis will also enable the attachment of covalent probes at specific, internal positions of annealed tRNA substrates for use in cross-linking and fluorescence studies. In this way, protein structural motifs and specific amino acid residues in close contact with specific regions of tRNA(Pro) will be identified. Several retroviruses contain transfer RNAs that prime viral DNA synthesis. For example, tRNA(Pro) is the specific primer for Moloney Murine leukemia virus DNA synthesis, and tRNA(Lys) is used to prime human immunodeficiency virus reverse transcriptase. This proposed investigation will provide insight into protein-RNA interactions which are important in the assembly and mechanism of oncogenic retroviruses.
