Incorrect aminoacylations catalysed by E. coli valyl-tRNA synthetase

Abstract

Summary It is possible to produce incorrect aminoacylations on yeast or E. coli tRNA's by purified valyl-tRNA synthetase from E. coli when special experimental conditions are used. These conditions differ from the classical ones by a high [Mg ++ ]/[ATP] ratio, a more alkaline pH, a long reaction time, a high enzyme concentration and in some cases the addition of dimethylsulfoxide. Under these conditions valine can be charged by E. coli valyl-tRNA synthetase on several yeast tRNA's (tRNA Val , tRNA Thr , tRNA Ile , tRNA Phe , tRNA Ala and tRNA Met ) and on a few E. coli tRNA's (tRNA Val , tRNA Ala and tRNA Met ). Some of these incorrect aminoacylation reactions have been studied more carefully using purified yeast tRNA Ala and tRNA Phe . It has been shown that the K m 's of these reactions are only increased by a factor of 10, as compared to the value obtained in the normal aminoacylation of E. coli tRNA Val , whereas the V max 's decrease strongly by a factor of 1000. It has been observed that whatever the aminoacylation conditions used, only a fraction of purified tRNA can be charged with the incorrect aminoacid. The incomplete aminoacylation is not due to enzyme inactivation, as the reaction resumes if a new amount of tRNA is added. The incorrect aminoacylations can be related to structural changes either at the enzyme or at the tRNA level. As they are found only with a limited number of tRNA's it could be that they involve some structural analogies between the tRNA normally recognised by E. coli valyl-tRNA synthetase and those tRNA's which are abnormally recognised. Sequence analogies between tRNA's of known sequence, aminoacylated without dimethylsulfoxide, are found especially in the dihydrouracil stem, in some nucleotides of the dihydrouracil loop, in the terminal part of the aminoacid acceptor stem and in the extra-loop. Some of these structural analogies are not conserved when the sequences of the tRNA's aminoacylated in the presence of dimethylsulfoxide are compared, but the similarities at the level of the extra-loop are still found.