9513932 Horowitz The long-range goals of this research are to define structural features of transfer RNA essential for its function in protein synthesis. Focus will be on E. coli tRNA Val and its interaction with its cognate aminoacyl-tRNA synthetase. Methods of tRNA gene manipulation, site-directed mutagenesis, and in vitro transcription will be used to introduce modifications into E. coli tRNA Val and examine their effects on function, primarily by examining in vitro aminoacylation kinetics and polypeptide synthesis. a. Role of the universally conserved 3'-CCA sequence in tRNA function. Recent results have shown that E. coli tRNA Val can function actively in aminoacylation and polypeptide synthesis despite alterations in the universally conserved 3'-CCA end. To further explore the functional role of the 3'-CCA terminus the following will be done: (1) Functional groups in the 3' terminal sequence of tRNA essential for aminoacylation will be examined by site-specific incorporation of base analogs. (2) Role of 3'-CCA in misacylation and editing by valyl-tRNA synthetase will be investigated. (3) Role of 3' terminal nucleotides in polypeptide synthesis will be investigated. Although the variants of tRNA Val termination in 3' pyrimidines are readily aminoacylated, they function poorly in polypeptide synthesis. The step(s) of polypeptide synthesis inhibited by pyrimidine substituents at the 3' end of tRNA Val will be identified. (4) Ribosome recognition of the 3' end will be investigated using 3'-end mutants of tRNA. Val (5) Specific requirement for 3'-CCA in tRNAs other than tRNA Val will be examined. b. Recognition of E. coli tRNA Val by valyl-tRNA synthetase. The recognition of tRNA Val by its cognate synthetase will be further investigated and the synthetase itself will be further characterized. These studies will include the following: (1) Efforts will continue to define the complete set of sequence and structural elements essential for tRNA Val recognition by valyl-tRNA syn thetase. Features thus far identified as necessary for tRNA Val recognition will be inserted into other tRNAs to assess effects of sequence context on valine-accepting specificity and to identify possible negative determinants (antideterminants) that interfere with synthetase recognition. (2) Details of synthetase/tRNA interactions can best be resolved by structure determination of the complex. Large quantities of purified tRNA Val and valyl-tRNA synthetase are available and in collaboration with a crystallographer the structure of valyl-tRNA synthetase and its complexes will be determined. (3) The aminoacylation fidelity, hydrolytic editing, and other functions of an active truncated mutant of valyl-tRNA synthetase, which have recently been isolated, cloned, and overexpressed, will be characterized. %%% Transfer RNAs (tRNA) play an important role in determining the accuracy and efficiency of protein biosynthesis. These molecules effectively translate the sequence of 3-nucleotide codes determined by the genetic material into the linear incorporation of amino acids into proteins in their proper order. All tRNAs contain a universally conserved code (3'-CCA) at one end of the molecule which implicates a specific function. Interestingly, base changes within this sequence had no effect on protein synthesis. The proposed research will explore the functional role of the 3'-CCA sequence by evaluating the effects of altering chemical(functional) groups within this sequence on aminoacylation reaction kinetics as well as on recognition by the specific tRNA synthetase which mediates this reaction. The structure of the synthetase enzyme and its complexes with tRNA and other substrates will be obtained by X-ray crystallography. ***
