Effects of Manipulating tRNAs on Protein Synthesis in E. coli

The proposed research deals with in vivo studies of the role of charged and uncharged tRNA molecules in the efficiency, accuracy, and regulation of translational elongation. (1) Cells with two kinds of tRNA mutations, miaA and ts-tRNATRP, will be transformed with modified RF2 frameshift constructs and tests will be made of the efficiency of the programmed frameshifts. Effects on these frameshifts of overexpressing tRNATRP from a cloned plasmid gene will also be determined. (2) Both wild type and hyperaccurate Smp strains containing the modified RF2 frameshift constructs will be subjected to manipulation both of charged and uncharged tRNATRP. Effects of uncharged tRNA and the fraction of charged tRNA on these programmed frameshifts will be determined. (3) A specific peptide from the MS2 coat protein containing residue 32, encoded by a UGG (Trp) codon, will be isolated in order to determine (by radioactive lableing and peptide mapping) what amino acid(s) is inserted at this position during Trp limitation. (4) The effect of manipulating charged and uncharged tRNATRP levels on the frequency of substitutions at Trp codons during rate-limiiting elongation will be examined. (5) A vector system to test translational efficiency of low- versus high-usage codons will be placed in the hyperaccurate Smp strains in order to test whether fidelity conditions alter "translational polarity". %%% During cell growth, aging, starvation, and disease protein synthesis is modulated. Although many hypotheses have been developed about how such regulation is accomplished and what the cell gains through such regulation, very few studies have been designed to evaluate the regulation of synthesis at the level of translation in living cells. The studies proposed here take advantage of a unique genetic/biochemical system which permits control of the concentrations of specific tRNA molecules in living cells and evaluating the protein synthetic effects of varying these tRNA levels.