Phenylalanyl-tRNA Synthetase and Isoleucyl-tRNA ^Phe : A Possible Verification Mechanism for Aminoacyl-tRNA

Abstract

The synthesis of isoleucyl-tRNAPhe (Escherichia coli) proceeds at an appreciable rate under normal in vitro conditions in the presence of isoleucyl-tRNA synthetase (EC 6.1.1.5) from E. coli. The misacylated product is shown here to be hydrolyzed by highly purified phenylalanyl-tRNA synthetase from E. coli, with release of isoleucine and active tRNAPhe. Thus, phenylalanyltRNA synthetase possesses a previously unrecognized activity, which deacylates a mistakenly acylated tRNAPhe; the enzyme is inactive toward correctly matched aminoacyl tRNAs. Such a mechanism could serve to verify aminoacyl-tRNAs, deacylating those that are misacylated. Thts, a common generalization needs to be modified: an amino acid is not necessarily committed to a given (incorrect) anticodon when it is incorporated into aminoacyl-tRNA. It may be possible to correct it thereafter.I have shown previously that homogeneous isoleucyl-tRNA synthetase (EC 6.1.1.5) from Escherichia coli can catalyze the synthesis of Ile-tRNAPhe (E. coli). Such isoleucine residues would be incorporated into polypeptides in response to phenylalanine codons (1).Does this occur in vivo, and if not, how is it prevented? With Ile-tRNAPhe as a probe, two previously unnoted mechanisms for insuring the correctness of the catalog of aminoacyltRNAs have been demonstrated. This paper describes the first of these, a hydrolytic deacylation of Ile-tRNAPhe by purified phenylalanyl-tRNA synthetase. MATERIALS AND METHODSTransfer RNAs were, with one exception, purified isoacceptors. Ala-tRNA was synthesized from unfractionated tRNA (2): 0.53 nmol Ala/10 A260 tRNA. Purified Ile-tRNAIle (E. coli) has been described (3). The [3H]Phe-tRNAPhe was synthesized from (> 78% pure) tRNAPhe (E. coli) (4); the purified Phe-tRNA synthetase described below was used: the product carried 6.5 nmol of ['H]Phe/10 A20 tRNA. Absorbance is measured after dilution in 0.01 M NaOH. Other aminoacyl-tRNAs were synthesized by use of mixed synthetases from E. coli MRE 600 (5); the purified tRNAs are from the tRNA purification project at Oak Ridge (4). The acylated tRNAs, isolated from the aminoacylation reaction by phenol extraction followed by alcohol precipitation in the presence of 0.5 M NaCl, had the following activities per 10 A260 Ile-tRNAPhe was prepared under normal acylation conditions; purified Ile-tRNA synthetase (1) and 20% methanol were added: the two preparations of [14C]Ile-tRNAPhe used here had, per 10 A260 units, 2.7 nmol and 3.6 nmol Ile-tRNA. Phe-tRNA synthetase from E. coli B was purified by a modification of the method of Fangman and Neidhardt (6), which is itself a modification of the procedure of Conway, Lansford, and Shive (8). An additional step was added to the previous procedure (6): chromatography of the diethylaminoethyl (DEAE)-cellulose fraction on an hydroxylapatite (Biogel HTP, Biorad Laboratories) column (0.8 X 13 cm). This column was eluted with a linear gradient of 0.01-0.20 M potassium phosphate buffer, pH 6.8 (250 ml total volume), with 7 mM 2-mercaptoethanol and 10% gly...

Elucidation of tRNA-dependent editing by a class II tRNA synthetase and significance for cell viability

“…In the case of class II enzymes, such as AlaRS, little is known about editing. Early preliminary work on AlaRS, PheRS and LysRS demonstrated editing activities for these class II enzymes (Yarus, 1972; Tsui and Fersht, 1981; Jakubowski, 1997). More recent work on the class II ProRS and ThrRS elucidated editing by these enzymes as well (Beuning and Musier‐Forsyth, 2000; Dock‐Bregeon et al ., 2000; Wong et al ., 2002).…”

tRNA‐dependent amino acid discrimination by yeast seryl‐tRNA synthetase

“…The editing reactions of class II synthetases have been studied much less than those of class I. Class II phenylalanyl-tRNA synthetase (PheRS) specifically deacylates Ile-tRNA Phe [13], and alanyl-tRNA synthetase (AlaRS) has been shown to hydrolyze misactivated serine and glycine [14]. Escherichia coli lysyl-tRNA synthetase (LysRS) hydrolyzes misactivated homocysteine, homoserine, cysteine, threonine and alanine [15].…”

Severe oxidative stress induces protein mistranslation through impairment of an aminoacyl-tRNA synthetase editing site

“…For example, threonyl-tRNA synthetase (ThrRS) misactivates serine (Ser) (18). Such errors are further corrected by a proofreading activity named editing (18)(19)(20)(21)(22)(23)(24)(25)(26). A double-sieve model that suggested that the active and editing sites are structurally distinct (27) was later confirmed by biochemical and structural analyses (28,29).…”