Loss of the precision of translation and transcription may be a factor in aging and degenerative diseases such as cancer (the Orgel error Catastrophe Hypothesis). We find that spermine and spermidine markedly enhance both the RATE and PRECISION of aminoacyl-tRNA synthbsis. They suppress misacylation - e.g., formation of Phe-tRNAVal. We propose first to study systems other than the yeast-Phe system described in the attached JBC manuscript (in press) to see whether polyamine suppression of misacylation is a general phenomenon. By determining rates and equilibrtia of association of enzyme with tRNA as a function of temperature and reaction conditions (high [Mg2+] favoring misacylation and low [Mg2+] + polyamine suppressing misacylation) we hope to determine the entropies of association, (to E.S), conformational change (to E.S*) and activation to (E.S+). Techniques used will be Temperature Jump, Stop-Flow and steady state kinetic analysis. We expect to confirm preliminary experiments that the polyamine effect is general. Also to find that the enzyme combines with many tRNA's to form an E.S of high entropy. With cognate tRNA, E.S equilibrtates rapidly with a conformer, E.S*, of comparible free energy but much lower entropy. Because E.S* is strained (very low entropy) it is nearer the transition state E.S minus or plus and hence quickly converted to product. Under conditions where misacylation occurs (high Mg2+), non-cognate tRNA forms some E.S* and is aminoacylated. The research may 1) reveal further information on how precision in trnscription and translation is maintained; 2) give the biochemical detail for an important function of polyamines; and 3) provide examples of Jenck's Circe Effect - the use of potential binding energy to induce stress in E.S for the sake of rate enhancement.
NIH ExPorter
NIH ExPorter