Misacylation of tRNALys with noncognate amino acids by lysyl-tRNA synthetase.

Lysyl-tRNA synthetase (LysRS), a class II enzyme whose major function is to provide Lys-tRNALys for protein synthesis, also catalyzes aminoacylation of tRNALys with arginine, threonine, methionine, leucine, alanine, serine, and cysteine. The limited selectivity in the tRNA aminoacylation reaction appears to be due to inefficient editing of some amino acids (Met, Leu, Cys, Ala, Thr) by a pre-transfer mechanism or the absence of editing of other amino acids (Arg and Ser). Purified Arg-tRNALys, Thr-tRNALys, and Met-tRNALys were essentially not deacylated by LysRS, indicating that the enzyme does not possess a post-transfer editing mechanism. However, LysRS possesses an efficient pre-transfer editing mechanism which prevents misacylation of tRNALys with ornithine. A novel feature of this editing reaction is that ornithine lactam is formed by the facile cyclization of ornithyl adenylate.     

Shifted positioning of the anticodon nucleotide residues of amber suppressor tRNA species by Escherichia coli arginyl-tRNA synthetase.

Cytidine in the anticodon second position (position 35) and G or U in position 36 of tRNAArg are required for aminoacylation by arginyl-tRNA synthetase (ArgRS) from Escherichia coli. Nevertheless, an arginine-accepting amber suppressor tRNA with a CUA anticodon (FTOR1Delta26) exhibits suppression activity in vivo [McClain, W.H. & Foss, K. (1988) Science, 241, 1804-1807]. By an in vitro kinetic study with mutagenized tRNAs, we showed that the arginylation of FTOR1Delta26 involves C34 and U35, and that U35 can be replaced by G without affecting the activity. Thus, the positioning of the essential nucleotides for the arginylation is shifted to the 5' side, by one residue, in the suppressor tRNAArg. We found that the shifted positioning does not depend on the tRNA sequence outside the anticodon. Furthermore, by a genetic method, we isolated a mutant ArgRS that aminoacylates FTOR1Delta26 more efficiently than the wild-type ArgRS. The isolated mutant has mutations at two nonsurface amino-acid residues that interact with each other near the anticodon-binding site.     

Structural analyses on yeast tRNA(Tyr) and its complex with tyrosyl-tRNA synthetase by the use of hydroxyl radical 'footprinting'.

Hydroxyl radical, generated by reduction of hydrogen peroxide by Fe(II)-EDTA, was used to investigate the contact sites of yeast tRNA(Tyr) with its cognate tyrosyl-tRNA synthetase (TyrRS). Exposure of free tRNA(Tyr) to this reagent gave cleavage patterns consistent with the tertiary structure of yeast tRNA(Phe) established by X-ray crystallography. When the probing reaction was performed under the conditions which stabilized complex formation between tRNA(Tyr) and TyrRS, aminoacyl-stem region of the tRNA was protected from cleavage. This result supports our earlier finding that the information for binding to TyrRS would reside mainly in the aminoacyl-stem of tRNA(Tyr).     

Misacylation and editing by Escherichia coli valyl-tRNA synthetase: evidence for two tRNA binding sites.

Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids. The 3'-end of tRNA plays a vital role in promoting the tRNA-dependent editing reaction. Valine tRNA having the universally conserved 3'-terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS. As a result 3'-end tRNA(Val) mutants, particularly those with 3'-terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine. Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3'-pyrimidines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine. Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS. We also observe misacylation of wild-type and 3'-end mutants of tRNA(Val) with isoleucine. Valyl-tRNA synthetase does not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme. Misacylated mutant tRNAs as well as 3'-end-truncated tRNA(Val) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule. Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS.     

Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.     

Overproduction and purification of lysyl-tRNA synthetase encoded by the herC gene of E coli.

Lysyl-tRNA synthetases are synthesized from two distinct genes in E coli, lysS and lysU, but neither gene product has been purified distinctively by using overproducing systems. The lysS gene has been identified by a herC mutation which restores maintenance of the mutant ColE1 replicon. The herC gene product was overproduced by using a tac promoter fusion and purified to homogeneity. The purified HerC protein possesses a lysyl-tRNA synthetase activity as predicted by the sequence identity of herC to lysS. The procedure is useful for rapid mass-scale purification of lysyl-tRNA synthetase.     

Recognition of E coli tRNAArg by arginyl tRNA synthetase.

Escherichia coli tRNAArg was digested with ribonuclease T1 under restrictive conditions in order to dissect a minimum number of diester bonds. The number of diester bonds cleaved and their locations were determined by phosphorylation of the newly formed 5' hydroxyl groups with [32P] ATP and polynucleotide kinase. There was complete loss of aminoacylation of tRNAARg when two diester bonds were cleaved at the anticodon. However, this material retained the specific properties of synthetase recognition. Two fragments were derived by further digestion of this tRNA. One 19 nucleotide-long fragment derived from the 3' end of tRNAArg and another 18 nucleotide-long fragment derived from the 5' end of the molecule were required to maintain the properties of the specific recognition by the arginyl tRNA synthetase in the absence of the rest of the structure including the anticodon.     

Isolation and properties of a plasmid which expresses the E. coli Su+7 amber suppressor tRNA gene.

Summary The gene of the amber suppressor tRNA derived from tRNA^Try, Su⁺7, has been inserted into a col E₁-derived vehicle by selecting for its expression. Despite selection for a suppressor phenotype, and the plasmid's stable presence at ca. 180 copies/cell during balanced growth, the level of mature tRNA maintained by the gene is less than that of the normal haploid tRNA^Try locus in the bacterial chromosome. Transfer RNA genes, both the plasmid Su⁺7 gene and chromosomal tRNA's are expressed during inhibition of protein synthesis. During, e.g. chloramphenicol inhibition, Su^-7 and Su⁺7 tRNA can be elevated similarly in the plasmid-containing cell; Su⁺7 reaches levels of molecules/cell which ordinarily characterize a major tRNA.The recombinant plasmid, but not the cloning vehicle alone, has a more general effect on tRNA levels; accumulation of tRNA from three chromosomal tRNA loci including tRNA^Try, continues during extensive isoleucine limitation. The plasmid therefore contains a locus which probably alters the relaxedstringent circuit, whose effects is disseminated to at least 3 widely separated loci.     

Allosteric interaction of nucleotides and tRNA(ala) with E. coli alanyl-tRNA synthetase

Alanyl-tRNA synthetase, a dimeric class 2 aminoacyl-tRNA synthetase, activates glycine and serine at significant rates. An editing activity hydrolyzes Gly-tRNA(ala) and Ser-tRNA(ala) to ensure fidelity of aminoacylation. Analytical ultracentrifugation demonstrates that the enzyme is predominately a dimer in solution. ATP binding to full length enzyme (ARS875) and to an N-terminal construct (ARS461) is endothermic (ΔH = 3-4 kcal mol(-1)) with stoichiometries of 1:1 for ARS461 and 2:1 for full-length dimer. Binding of aminoacyl-adenylate analogues, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (ASAd) and 5'-O-[N-(L-glycinyl)sulfamoyl]adenosine (GSAd), are exothermic; ASAd exhibits a large negative heat capacity change (ΔC(p) = 0.48 kcal mol(-1) K(-1)). Modification of alanyl-tRNA synthetase with periodate-oxidized tRNA(ala) (otRNA(ala)) generates multiple, covalent, enzyme-tRNA(ala) products. The distribution of these products is altered by ATP, ATP and alanine, and aminoacyl-adenylate analogues (ASAd and GSAd). Alanyl-tRNA synthetase was modified with otRNA(ala), and tRNA-peptides from tryptic digests were purified by ion exchange chromatography. Six peptides linked through a cyclic dehydromoropholino structure at the 3'-end of tRNA(ala) were sequenced by mass spectrometry. One site lies in the N-terminal adenylate synthesis domain (residue 74), two lie in the opening to the editing site (residues 526 and 585), and three (residues 637, 639, and 648) lie on the back side of the editing domain. At least one additional modification site was inferred from analysis of modification of ARS461. The location of the sites modified by otRNA(ala) suggests that there are multiple modes of interaction of tRNA(ala) with the enzyme, whose distribution is influenced by occupation of the ATP binding site.     

Interaction of the tRNA(Phe) acceptor end with the synthetase involves a sequence common to yeast and Escherichia coli phenylalanyl-tRNA synthetases

Modified lysines resulting from the cross-linking of the 3' end of tRNA(Phe) to yeast phenylalanyl-tRNA synthetase (an enzyme with an alpha 2 beta 2 structure) have been characterized by sequencing the labeled chymotryptic peptides that were isolated by means of gel filtration and reversed-phase chromatography. The analysis showed that Lys131 and Lys436 in the alpha subunit are the target sites of periodate-oxidized tRNA(Phe). Mutant protein with a Lys----Asn substitution established that each lysine contributes to the binding of the tRNA but is not essential for catalysis. The major labeled lysine (K131) belongs to the sequence IALQDKL (residues 126-132), which shares three identities with the peptide sequence ADKL found around the tRNAox-labeled Lys61 in the large subunit of Escherichia coli phenylalanyl-tRNA synthetase [Hountondji, C., Schmitter, J. M., Beauvallet, C., & Blanquet, S. (1987) Biochemistry 26, 5433-5439].