Prof. Sidney Hecht, University of Virginia

Protein Synthesis Using Tandemly Activated tRNAs

Sidney M. Hecht
Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia

While the occurrence of bisphenylalanyl-tRNAs have been reported previously in a thermophilic bacterium, the ability of bisaminoacylated transfer RNAs to function as an amino acid donor or acceptor in protein synthesis has not been explored.

Presently, we describe the preparation of bisaminoacylated tRNAs by our "chemical aminoacylation" procedure, including the facility of bisacylation as a function of amino acid structure and strategies for preparing bisaminoacylated tRNAs having different amino acids on the 2' and 3'-positions of the 3'-terminal adenosine moiety. For such differentially substituted tRNAs, the assignment of regiochemistry of substitution has also been realized. We report the use of tandemly activated tRNAs in protein synthesis (in direct comparison with monoaminoacylated tRNAs) in both prokaryotic and eukaryotic protein synthesizing systems. Further, it is demonstrated that the tandemly aminoacylated species exhibit surprising chemical and biochemical stability over a range of pH values and temperatures, consistent with their possible utilization in organisms such as thermophilic bacteria. Unequivocal evidence is provided that the first amino acid incorporated into protein from the bisaminoacylated tRNA is the amino acid attached to the 3'-position of the terminal adenosine moiety of the tRNA, implying that acceptance of the growing polypeptide chain during protein synthesis by the tRNA in the ribosomal A-site normally involves the positioning of the aminoacyl moiety at the 3'-position of that tRNA. Likewise, following translocation the extended peptide chain is donated from the 3'-position of the peptidyl-tRNA in the ribosomal P-site.

For more information contact Dr. Adegboyega Oyelere (404-894-4047).