Threonyl‐tRNA synthetase from yeast Aminoacylation of tRNA on its non‐accepting 3′‐terminal hydroxyl group and its behaviour in enzyme‐catalyzed deacylation

Abstract

Methods have been developed by which tRNAThr may be aminoacylated at the normally non–accepting 3’–terminal ribose OH. Two of the methods utilize the mischarging ability of the synthetases under special conditions of low salt concentration and presence of organic solvents. The third method demonstrates for the first time that for some synthetases the 2’,3’ specificity may be manipulated by use of similar special conditions. In the case of threonyl–tRNA synthetase, Thr–tRNAThr–C–C–A(3’d) has been synthesised by this method. The behaviour of threonyl esters of tRNAThr–C–C–A, tRNAThr–C–C–A(2’d) and tRNAThr–C–C–A(3’d) in the free enzyme–catalyzed deacylation has been studied and the results indicate that the cis diol functional group is necessary for this hydrolysis. The position on the terminal ribose from which the amino acid is removed in this reaction remains to be identified.

Mechanism of tRNA-dependent editing in translational quality control

“…On the other hand, in the proposed ThrRS model, two water molecules hydrogen-bonded by editing site residues were proposed to mediate substrate hydrolysis (18), which is also analogous to PheRS, although contributions of individual ThrRS editing site residues have not yet been quantified. Reexamination of the E. coli ThrRS structure revealed that the 2Ј-OH of A76 lies close to one catalytic water, and the recent crystal structure of Pyrococcus abyssi ThrRS revealed that the 2Ј-OH indeed interacts with a candidate catalytic water molecule (38), raising the intriguing possibility that the 2Ј-OH of tRNA may contribute to ThrRS editing in much the same way the 3Ј-OH assists PheRS (39). The high similarity in the editing mechanisms of PheRS and ThrRS, both class II aaRSs, would not seem to extend to class I aaRSs such as IleRS and LeuRS, which may partly reflect the general observation that the rate-determining step differs between the two enzyme classes (40).…”

Review

“…The molecular ratios of synthetases to ribosomes and elongation factor EF-Tu appeared to be at balance at different growth rates. In most cases the preparation procedures follow the classical ways of protein isolation including precipitation by ammonium sulfate, adsorption by hydroxylapatite, calcium phosphate, or alumina Cy gels, column chromatography on hydroxylapatite, DEAE-cellulose, DEAE-Sephadex, or Cibacron Allende eta/., 1966Dignam et a/., 1980Ogata ef a/., 1994Hirsh, 1968Hennecke ef a/., 1977Kern and Lapointe, 1979Anderson and Fowden, 1970Grosjeanefa/,,1976Roberts and Olsen, 1976lgloiefa/,,1977Igloi and Cramer, 1978von der Haar and Cramer, 1978Freist ef a/., 1978Yamada, 1978aYamada, 1978bPanefa/.,1982a,b Fedorovefa/,,1986Tagaefa/.,1986Yaremchukefa/,,1989 Garber ef a/., 1990 Zheltonosova ef a/., 1994 Kumazawa ef a/., 1991Berg, 1993 Blue gel (Table 1). In most cases the preparation procedures follow the classical ways of protein isolation including precipitation by ammonium sulfate, adsorption by hydroxylapatite, calcium phosphate, or alumina Cy gels, column chromatography on hydroxylapatite, DEAE-cellulose, DEAE-Sephadex, or Cibacron Allende eta/., 1966Dignam et a/., 1980Ogata ef a/., 1994Hirsh, 1968Hennecke ef a/., 1977Kern and Lapointe, 1979Anderson and Fowden, 1970Grosjeanefa/,,1976Roberts and Olsen, 1976lgloiefa/,,1977Igloi and Cramer, 1978von der Haar and Cramer, 1978Freist ef a/., 1978Yamada, 1978aYamada, 1978bPanefa/.,1982a…”