THE EFFECT OF ALTERED ISOLEUCINE-VALINE BIOSYNTHESIS ON THE BRANCHED-CHAIN AMINOACYL-TRANSFER-RNA SYNTHETASES IN ESCHERICHIA COLI K-12
Abstract
The synthesis of the branched-chain aminoacyl-tRNA synthetases responds to the supply of the cognate amino acids in the cell. In addition, a mutation in the ilvA gene, specifying threonine deaminase, the first enzyme in the biosynthesis of isoleucine, results in lower levels of the isoleucyl-, valyl-, and leucyl-tRNA synthetases. I present evidence for a physical interaction between threonine deaminase, (alpha)-ketobutyrate, and isoleucyl-tRNA synthetase by gel filtration of these three macromolecules on Sephadex-G150. Furthermore, the enzyme activity of purified isoleucyl-tRNA synthetase is inhibited by the presence of purified threonine deaminase and (alpha)-ketobutyrate in the reaction mixture. I propose a model to explain the effect of mutations in the ilvA gene on the expression of the ileS and valS genes, specifying isoleucyl- and valyl-tRNA synthetase, respectively. The formation of a complex between isoleucyl- or valyl-tRNA synthetase, threonine deaminase, and (alpha)-ketobutyrate lowers the activities of the synthetases, and less of the reaction products, isoleucine-tRNA('Ile) and valine-tRNA('Val), is formed. A proposed mechanism requiring charged tRNA for attenuation of ileS and valS gene expression predicts increased expression of ileS and valS when levels of isoleucine-tRNA('Ile) and valine-tRNA('Val) are reduced. This model explains the following results: (1) (alpha)-Ketobutyrate inhibits the growth of strain PS1150 (ilvA538) but not PS1079 (ilvA('+)). The valyl- and isoleucyl-tRNA synthetases are elevated three fold in PS1150 grown in glucose minimal medium plus (alpha)-ketobutyrate. No increase in synthetase activity was observed in PS1079 under similar growth conditions. (2) Strain PS1150 (ilvA538) has two-fold lower valyl-, isoleucyl-, and leucyl- tRNA synthetase activities than strain PS1079 (ilvA('+)). A revertant of PS1150 that has wild-type levels of valyl- and isoleucyl- tRNA synthetase carries a mutation in the ilvH gene, designating a valine-resistant acetohydroxyacid synthase III. This strain has high intracellular pools of valine. Valine activates threonine deaminase, and an increase in the product, (alpha)-ketobutyrate, increases the valyl- and isoleucyl-tRNA synthetase as predicted in the model. (3) Strain NP29 (valS(ts)) has a lower than normal valyl-tRNA synthetase activity, while the steady state level of valyl- and isoleucyl-tRNA synthetase, as measured by two-dimensional gel electrophoresis and the attachment of the ('14)C-amino acid to transfer RNA, is two-fold higher in NP29 (valS(ts)) than in NP2 (valS('+)). (4) Crude dialyzed extracts of PS1150 (ilvA538) contain a valyl-tRNA synthetase activity inhibited by (alpha)-ketobutyrate, in vitro eight times greater than in PS1079 (ilvA('+)), and an isoleucyl-tRNA synthetase activity inhibited three times greater than in PS1079.
Degree
Ph.D.
Subject Area
Microbiology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.