The reliability of in vivo structure-function analysis of tRNA aminoacylation

The G.U wobble base-pair in the acceptor helix of Escherichia coli tRNA(Ala) is critical for aminoacylation by the alanine synthetase. Previous work by several groups probed the mechanism of enzyme recognition of G.U by a structure-function analysis of mutant tRNAs using either a cell assay (amber suppressor tRNA) or a test tube assay (phage T7 tRNA substrate and purified enzyme). However, the aminoacylation capacity of particular mutant tRNAs was about 10(4)-fold higher in the cell assay. This led us to scrutinize the cell assay to determine if any parameter exaggerates the extent of aminoacylation in mutants forming substantial amounts of alanyl-tRNA(Ala). Ln doing so, we have refined and developed experimental designs to analyze tRNA function. We examined the level of aminoacylation of amber suppressor tRNA(Ala) with respect to the method of isolating aminoacyl-tRNA, the rate of cell growth, the cellular levels of alanine synthetase and elongation factor TU (EF-Tu), the amount of tRNA and the characteristics of EF-Tu binding. Within the precision of our measurements, none of these parameters varied in a way that could significantly amplify cellular alanyl-tRNA(Ala). A key observation is that the extent of aminoacylation of tRNA(Ala) was independent of tRNA(Ala) concentration over a 75-fold range. Therefore, the cellular assay of tRNA(Ala) reflects the substrate quality of the molecule for formation of alanyl-tRNA(Ala). These experiments support the authenticity of the cellular assay and imply that a condition or factor present in the cell assay may be absent in the test tube assay. (C) 1999 Academic Press.