Met-tRNA synthetase

The distinction between an initiating (5 )AUG and an internal one is straightforward. In bacteria, the two types of tRNA specific for methionine are designated tRNAMet and tRNAfMet. The amino acid incorporated in response to the (5 )AUG initiation codon is A7-formyl-methionine (fMet). It arrives at the ribosome as A7-formylmethionyl-tRNAfMet (fMet-tRNAfMet), which is formed in two successive reactions. First, methionine is attached to tRNAfMet by the Met-tRNA synthetase (which in E. coli aminoacylates both tRNAfMet and tRNAMet) ... 

The transformylase is more selective than the Met-tRNA synthetase it is specific for Met residues attached to tRNAfMet, presumably recognizing some unique structural feature of that tRNA. By contrast, Met-tRNAMet inserts methionine in interior positions in polypeptides. 

Cech s group was the first to have success in this direction (Piccirilli, 1992). Using a genetically modified Tetrahymena ribozyme, they were able to hydrolyse an ester bond between the amino acid A-formylmethionine and the corresponding tRNAf Met. The reaction was, however, very slow, only about 5 to 15 times faster than the uncatalysed reaction. The authors ventured to suggest that these ribozymes could have functioned as the first aminoacyl tRNA synthetases. 

The structure of the isoleucyl-tRNA synthetase (IleRS) from Thermus ther-mophilus (1045 residues, Mr 120 000) has been solved, as well as its complexes with lie and Val.17 The protein contains a nucleotide binding fold (Chapter 1) that binds ATR The fold has two characteristic ATP binding motifs His-54-Val-55-Gly-56-His-57 and Lys-591-Met-592-Ser-593-Lys-594. In the L-Ile-IleRS complex, a single He is bound at the bottom of the ATP cleft, with the hydrophobic side chain in a hydrophobic pocket, surrounded by Pro-46, Trp-518, and Trp-558. L-Leucine cannot fit into this pocket because of the steric hindrance of one of its terminal methyl groups. Larger amino acids are similarly excluded from this site. In the l-Val-IleRS complex, Val is bound to the same site, but the... 

Ribosomes needed for translation in the PURE system are isolated from E. coli using sucrose-density gradient centrifugation. The protein factors necessary for translation in E. coli are recombinantly expressed as His-tagged fusions, and purified to homogeneity. These include the factors for initiation (IFl, IF2, and IF3), elongation (EF-G, FF-Tu, FF-Ts), peptide chain release (RFl and RF3), ribosome recycling (RRF), methionyl-tRNA transformylase (MTF) for formylation of the initial Met-tRNA, and the 20 aminoacyl-tRNA synthetases (ARSs) for transfer RNA (tRNA) recy-... 

The nucleotidyl transfer step is reaction (29a), which proceeds with inversion of configuration at phosphorus in all of the aminoacyl-tRNA synthetase reactions so far studied [for amino acids (aa) Phe, He, Tyr, and Met] (89-92). Stereochemical inversion shows that the nucleotidyl transfer mechanism involves an uneven number of substitutions on phosphorus. Since no other evidence of an adenylyl-enzyme can be found, aminoacyl activation most likely occurs by a single-displacement mechanism, with direct transfer of the AMP group from ATP to the carboxylate group of the amino acid within the enzyme-amino acid-ATP complex. 

Fig. 12. P-NMR spectra (at 145.7 MHz) of(A) an equilibrium mixture initiated by mixing MgATP, methionine, and modified methionyl-tRNA synthetase (K [E-AMP-Met MgPPJ/[E Met MgATP]) with [E] > [ATP] at 20 C and pH 7.6 (B) after saturating the ) P(ATP) resonance by radio-frequency irradiation and (C) as in (B), but with added excess EDTA. Shifts upheld are negative. From Fayat et at. (1980).

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