tRNA recognition site of Escherichia coli methionyl-tRNA synthetase

We have previously shown that anticodon bases are essential for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase (MetRS) [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759] and that the enzyme tightly binds to C34 at the wobble position of E. coli initiator methionine tRNA (tRNAfMet) [Pelka, H., & Schulman, L. H. (1986) Biochemistry 25, 4450-4456]. We have also previously demonstrated that an affinity labeling derivative of tRNAfMet can be quantitatively cross-linked to the tRNA binding site of MetRS [Valenzuela, D., & Schulman, L. H. (1986) Biochemistry 25, 4555-4561]. Here, we have determined the site in MetRS which is cross-linked to the anticodon of tRNAfMet, as well as the location of four additional cross-links. Only a single peptide, containing Lys465, is covalently coupled to C34, indicating that the recognition site for the anticodon is close to this sequence in the three-dimensional structure of MetRS. The D loop at one corner of the tRNA molecule is cross-linked to three peptides, containing Lys402, Lys439, and Lys596. The 5' terminus of the tRNA is cross-linked to Lys640, near the carboxy terminus of the enzyme. Since the 3' end of tRNAfMet is positioned close to the active site in the N-terminal domain [Hountondji, C., Blanquet, S., & Lederer, F. (1985) Biochemistry 24, 1175-1180], this result indicates that the carboxy ends of the two polypeptide chains of native dimeric MetRS are folded back toward the N-terminal domain of each subunit.     

Identification of peptide sequences at the tRNA binding site of Escherichia coli methionyl-tRNA synthetase

Four different structural regions of Escherichia coli tRNAfMet have been covalently coupled to E. coli methionyl-tRNA synthetase (MetRS) by using a tRNA derivative carrying a lysine-reactive cross-linker. We have previously shown that this cross-linking occurs at the tRNA binding site of the enzyme and involves reaction of only a small number of the potentially available lysine residues in the protein [Schulman, L. H., Valenzuela, D., & Pelka, H. (1981) Biochemistry 20, 6018-6023; Valenzuela, D., Leon, O., & Schulman, L. H. (1984) Biochem. Biophys. Res. Commun. 119, 677-684]. In this work, four of the cross-linked peptides have been identified. The tRNA-protein cross-linked complex was digested with trypsin, and the peptides attached to the tRNA were separated from the bulk of the tryptic peptides by anion-exchange chromatography. The tRNA-bound peptides were released by cleavage of the disulfide bond of the cross-linker and separated by reverse-phase high-pressure liquid chromatography, yielding five major peaks. Amino acid analysis indicated that four of these peaks contained single peptides. Sequence analysis showed that the peptides were cross-linked to tRNAfMet through lysine residues 402, 439, 465, and 640 in the primary sequence of MetRS. Binding of the tRNA therefore involves interactions with the carboxyl-terminal half of MetRS, while X-ray crystallographic data have shown the ATP binding site to be located in the N-terminal domain of the protein [Zelwer, C., Risler, J. L., & Brunie, S. (1982) J. Mol. Biol. 155, 63-81].(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of the anticodon domain of tRNA(fMet) to Escherichia coli methionyl-tRNA synthetase

A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro. This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2). The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence. Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).     

Structural basis for anticodon recognition by methionyl-tRNA synthetase

In the 2.7-A resolution crystal structure of methionyl-tRNA synthetase (MetRS) in complex with tRNA(Met) and a methionyl-adenylate analog, the tRNA anticodon loop is distorted to form a triple-base stack comprising C34, A35 and A38. A tryptophan residue stacks on C34 to extend the triple-base stack. In addition, C34 forms Watson-Crick-type hydrogen bonds with Arg357. This structure resolves the longstanding question of how MetRS specifically recognizes tRNA(Met).     

Identification of potential amino acid residues supporting anticodon recognition in yeast methionyl-tRNA synthetase

Sequence comparisons among methionyl-tRNA synthetases from different organisms reveal only one block of homology beyond the last beta strand of the mononucleotide fold. We have introduced a series of semi-conservative amino acid replacements in the conserved motif of yeast methionyl-tRNA synthetase. The results indicate that replacements of two polar residues (Asn584 and Arg588) affected specifically the aminoacylation reaction. The location of these residues in the tertiary structure of the enzyme is compatible with a direct interaction of the amino acid side-chains with the tRNA anticodon.     

Identification of an amino acid region supporting specific methionyl-tRNA synthetase: tRNA recognition

Site-directed nuclease digestion and nonsense mutations of the Escherichia coli metG gene were used to produce a series of C-terminal truncated methionyl-tRNA synthetases. Genetic complementation studies and characterization of the truncated enzymes establish that the methionyl-tRNA synthetase polypeptide (676 residues) can be reduced to 547 residues without significant effect on either the activity or the stability of the enzyme. The truncated enzyme (M547) appears to be similar to a previously described fully active monomeric from of 64,000 Mr derived from the native homodimeric methionyl-tRNA synthetase (2 x 76,000 Mr) by limited trypsinolysis in vitro. According to the crystallographic three-dimensional structure at 2.5 A resolution of this trypsin-modified enzyme, the polypeptide backbone folds into two domains. The former, the N-domain, contain a crevice that is believed to bind ATP. The latter, the C-domain, has a 28 C-residue extension (520 to 547), which folds back, toward the N-domain and forms an arm linking the two domains. This study shows that upon progressive shortening of this C-terminal extension, the enzyme thermostability decreases. This observation, combined with the study of several point mutations, allows us to propose that the link made by the C-terminal arm of M547 between its N and C-terminal domains is essential to sustain an active enzyme conformation. Moreover, directing point mutations in the 528-533 region, which overhangs the putative ATP-binding site, demonstrates that this part of the C-terminal arm participates also in the specific complexation of methionyl-tRNA synthetase with its cognate tRNAs.     

Activation of methionine by Escherichia coli methionyl-tRNA synthetase

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of residues involved in the binding of methionine by Escherichia coli methionyl-tRNA synthetase

Comparison of the amino-acid sequences of several methionyl-tRNA synthetases indicates the occurrence of a few conserved motifs, having a possible functional significance. The role of one of these motifs, centered at position 300 in the E. coli enzyme sequence, was assayed by the use of site-directed mutagenesis. Substitution of the His301 or Trp305 residues by Ala resulted in a large decrease in methionine affinity, whereas the change of Val298 into Ala had only a moderate effect. The catalytic rate of the enzyme was unimpaired by these substitutions. It is concluded that the above conserved amino-acid region is located at or close to the amino-acid binding pocket of methionyl-tRNA synthetase.     

Peptides at the tRNA binding site of the crystallizable monomeric form of E. coli methionyl-tRNA synthetase.

A protein affinity labeling derivative of E. coli tRNA(fMet) carrying lysine-reactive cross-linking groups has been covalently coupled to monomeric trypsin-modified E. coli methionyl-tRNA synthetase. The cross-linked tRNA-synthetase complex has been isolated by gel filtration, digested with trypsin, and the tRNA-bound peptides separated from the bulk of the free tryptic peptides by anion exchange chromatography. The bound peptides were released from the tRNA by cleavage of the disulfide bond of the cross-linker and purified by reverse-phase high-pressure liquid chromatography, yielding three major peptides. These peptides were found to cochromatograph with three peptides of known sequence previously cross-linked to native methionyl-tRNA synthetase through lysine residues 402, 439 and 465. These results show that identical lysine residues are in close proximity to tRNA(fMet) bound to native dimeric methionyl-tRNA synthetase and to the crystallizable monomeric form of the enzyme, and indicate that cross-linking to the dimeric protein occurs on the occupied subunit of the 1:1 tRNA-synthetase complex.     

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the alkylation of the exocyclic amine of A37 by a dimethylallyl unit in tRNAs with an adenosine in the third anticodon position (position 36). By use of purified recombinant enzyme, steady- state kinetic studies were conducted with chemically synthesized RNA oligoribonucleotides to determine the essential elements within the tRNA anticodon stem-loop structure required for recognition by the enzyme. A 17-base oligoribonucleotide corresponding to the anticodon stem-loop of E. coli tRNA(Phe) formed a stem-loop minihelix (minihelix(Phe)) when annealed rapidly on ice, while the same molecule formed a duplex structure with a central loop when annealed slowly at higher concentrations. Both the minihelix and duplex structures gave k(cat)s similar to that for the normal substrate (full-length tRNA(Phe) unmodified at A37), although the K(m) for minihelix(Phe) was approximately 180-fold higher than full-length tRNA. The A36-A37-A38 motif, which is completely conserved in tRNAs modified by the enzyme, was found to be important for modification. Changing A36 to G in the minihelix resulted in a 260-fold reduction in k(cat) compared to minihelix(Phe) and a 13-fold increase in K(m). An A38G variant was modified with a 9-fold reduction in k(cat) and a 5-fold increase in K(m). A random coil 17-base oligoribonucleotide in which the loop sequence of E. coli tRNA(Phe) was preserved, but the 5 base pair helix stem was completely disrupted and showed no measurable activity, indicating that a helix-loop structure is essential for recognition. Finally, altering the identity of several base pairs in the helical stem did not have a major effect on catalytic efficiency, suggesting that the enzyme does not make base-specific contacts important for binding or catalysis in this region.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

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.     

Crystal structure of Escherichia coli methionyl-tRNA synthetase highlights species-specific features

The 3D structure of monomeric C-truncated Escherichia coli methionyl-tRNA synthetase, a class 1 aminoacyl-tRNA synthetase, has been solved at 2.0 A resolution. Remarkably, the polypeptide connecting the two halves of the Rossmann fold exposes two identical knuckles related by a 2-fold axis but with zinc in the distal knuckle only. Examination of available MetRS orthologs reveals four classes according to the number and zinc content of the putative knuckles. Extreme cases are exemplified by the MetRS of eucaryotic or archaeal origin, where two knuckles and two metal ions are expected, and by the mitochondrial enzymes, which are predicted to have one knuckle without metal ion.     

Covalent coupling of 4-thiouridine in the initiator methionine tRNA to specific lysine residues in Escherichia coli methionyl-tRNA synthetase

A new method has been developed to couple a lysine-reactive cross-linker to the 4-thiouridine residue at position 8 in the primary structure of the Escherichia coli initiator methionine tRNA (tRNAfMet). Incubation of the affinity-labeling tRNAfMet derivative with E. coli methionyl-tRNA synthetase (MetRS) yielded a covalent complex of the protein and nucleic acid and resulted in loss of amino acid acceptor activity of the enzyme. A stoichiometric relationship (1:1) was observed between the amount of cross-linked tRNA and the amount of enzyme inactivated. Cross-linking was effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAPhe. The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were purified from excess free peptides by anion-exchange chromatography. The tRNA was then degraded with T1 ribonuclease, and the peptides bound to the 4-thiouridine-containing dinucleotide were purified by high-pressure liquid chromatography. Two major peptide products were isolated plus several minor peptides. N-Terminal sequencing of the peptides obtained in highest yield revealed that the 4-thiouridine was cross-linked to lysine residues 402 and 439 in the primary sequence of MetRS. Since many prokaryotic tRNAs contain 4-thiouridine, the procedures described here should prove useful for identification of peptide sequences near this modified base when a variety of tRNAs are bound to specific proteins.