Isoleucyl‐tRNA synthetase from baker's yeast: The 3′‐hydroxyl group of the 3′‐terminal ribose is essential for preventing misacylation of tRNA<sup>Ile</sup>‐C‐C‐A with misactivated valine

Haar, Friedrich von der; Cramer, Friedrich

doi:10.1016/0014-5793(75)81094-5

Cited by 43 publications

(15 citation statements)

References 10 publications

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“…tRNA1"-C-C-3'dA can be easily acylated with the non-cognate amino acid valine, whereas the natural substrate tRNA"'-C-C-A is practically not charged with this amino acid [4]. Thus, it was concluded that 3'-OH must be essential for preventing misacylation and a mechanism was proposed in which the non-accepting hydroxyl mediates the nucleophilic attack of a water molecule on the ester linkage of misacylated tRNA [5].…”

Section: E-he-amp + -E+amp+ Ho Oh Ho 0-llementioning

confidence: 99%

Isoleucyl‐tRNA synthetase from baker's yeast

Freist

Sternbach²

1989

European Journal of Biochemistry

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Specificity with regard to amino acids in aminoacylation of tRNAIle‐C‐C‐3′dA by isoleucyl‐tRNA synthetase is characterized by discrimination factors (D2) which are calculated from kcat and Km values. The lowest values are observed for Cys, Val, His, and Trp (D2= 180–1700), indicating that at same amino acid concentrations isoleucine is 180–1700 times more attached to tRNAIle‐C‐C‐3′dA. The highest values are observed for Gly, Ala, Ser, Pro, Gln, Leu, Glu, and Phe (D2= 10 000–30 000). D2 values of the other amino acids are in the range of 2000–10 000. Recognition of most amino acids is achieved in a four‐step process. Two initial discrimination steps are due to different hydrophobic interactions with the binding pockets; two proof‐reading steps occur on the pre‐ and the post‐transfer stage. For nine amino acids (Ser, Asp, Asn, Val, Leu, His, Phe, Lys, Trp) post‐transfer proof‐reading is negligible. As a special case in discrimination of valine, one initial discrimination step and the post‐transfer proof‐reading step are lacking. The role of the terminal hydroxyl groups of the tRNA for post‐transfer proof‐reading is assigned to a simple neighbouring group effect. No preference for the 2′ or 3′ position in proof‐reading can be postulated.

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Section: E-he-amp + -E+amp+ Ho Oh Ho 0-llementioning

confidence: 99%

Isoleucyl‐tRNA synthetase from baker's yeast

Freist

Sternbach²

1989

European Journal of Biochemistry

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“…Early work demonstrated an important role for the 3Ј-end of tRNA Ile and tRNA Val in editing (17)(18)(19). However, the exact nature of this role was not determined.…”

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confidence: 99%

Plasticity of Recognition of the 3′-End of Mischarged tRNA by Class I Aminoacyl-tRNA Synthetases

Nordin

Schimmel

2002

Journal of Biological Chemistry

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Certain aminoacyl-tRNA synthetases prevent potential errors in protein synthesis through deacylation of mischarged tRNAs. For example, the close homologs isoleucyl-tRNA synthetase (IleRS) and valyl-tRNA synthetase (ValRS) deacylate Val-tRNA Ile and Thr-tRNA Val , respectively. Here we examined the chemical requirements at the 3-end of the tRNA for these hydrolysis reactions. Single atom substitutions at the 2-and 3-hydroxyls of a variety of mischarged RNAs revealed that, while acylation is at the 2-OH for both enzymes, IleRS catalyzes deacylation specifically from the 3-OH and not from the 2-OH. In contrast, ValRS can deacylate non-cognate amino acids from the 2-OH. Moreover, for IleRS the specificity for a 3-O location of the scissile ester bond could be forced to the 2-position by introduction of a 3-O-methyl moiety. Cumulatively, these and other results suggest that the editing sites of these class I aminoacyl-tRNA synthetases have a degree of inherent plasticity for substrate recognition. The ability to adapt to subtle differences in mischarged RNAs may be important for the high accuracy of aminoacylation.The genetic code is based on the accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetases (1, 2). These enzymes synthesize aminoacyl-tRNA in two steps. The amino acid is first reacted with ATP to give an activated aminoacyl adenylate, and then transesterified to the 3Ј-end of the tRNA. Aminoacyl-tRNA synthetases must precisely recognize both amino acid and tRNA substrates to yield the correct product. While the structural diversity of tRNA molecules allows for rigorous selection based on RNA-protein interactions, differentiating between closely related amino acids is more challenging. Years ago, Pauling (3) noted the intrinsic difficulty for isoleucyl-tRNA synthetase in the recognition of isoleucine over valine through simple binding interactions.Valine, which differs from isoleucine by a single methylene unit, is activated by Escherichia coli IleRS 1 only 180-fold less efficiently than isoleucine (4). However, the substitution of valine for isoleucine at isoleucine codons in the cell is less than 1 in 3000 (5). The increased specificity is a result of the RNAdependent editing of misactivated valine by IleRS (6, 7). A highly related class I aminoacyl-tRNA synthetase, valyl-tRNA synthetase, faces a similar dilemma in the accurate aminoacylation of tRNA Val . Threonine, an isostere of valine, is activated at a rate 250-fold reduced from that of valine (8). Like IleRS, ValRS prevents the misincorporation of threonine into proteins through the RNA-dependent editing of misactivated threonine (9).These reactions strictly require the presence of the cognate tRNA (6, 10). In the absence of tRNA, the enzymatically generated misactivated adenylates remain in the active site, sequestered from hydrolysis. Upon addition of cognate tRNA the misactivated amino acids are hydrolyzed, regenerating the free tRNA and amino acid, while converting 1 equivalent of ATP to AMP. A prominent mechanism for editing misactivated a...

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“…The aminoacylation mixture consisted of 360 ,ul (total volume) of 13 mM NH4+-Pipes buffer [ammopium piperazine-N,N'-bis-(2-ethanesulfonate)], pH 7.0, containing 0.13 M KC1, 20 mM MgCl2, 0.7 mM EDTA, 1.5-8 ,uCi of 3H-labeled amino acid at 2-10 MM concentration, 8 MM of'each of 18 other unlabeled amino acids (except cysteine) to prevent misacylations (24), 2 mM ATP, and 0.8-1.6 A2(0 units of unfractionated tRNA species la, 2a, or 3a. The reaction was initiated by the addition of 30 Ml of E. coli aminoacyl-tRNA synthetase solution and maintained at room temperature for 30 (tRNA species la) or 60 (2a and 3a) min.…”

Section: Methodsmentioning

confidence: 99%

Isomeric aminoacyl-tRNAs are both bound by elongation factor Tu.

Hecht

Tan

Chinault

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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Recent suggestions that elongation factor Tu (EF-Tu) is specific for 2'-0-aminoacyl-tRNA, as compared with the 3'-isomer, prompted us to assay [3Hlaminoacyl-tRNAs from Escherichia coli terminating in 2'-or 3'-deoxyadenosine for binding to EF-Tu to determine the possible positional specificity of the factor. Binding of modified aminaoc l-tRNAs to EFTuGTP was measured both as a function of the ability of EFTuGTP to diminish the rate of chemical deacylation of[3H]aminoacyl-tRNAs and by gel filtration of the individual ternary complexes. Fifteen different tRNA isoacceptors were tested by the deacylation procedure, including three (tRNAAsP, tRNACYs, and tRNATYr) for which isomeric modified aminoacyl-tRNAs were available. All of the modified aminoacyltRNAs were protected from deacylation, although generally to a lesser extent than the corresponding unmodified species.Six modified tRNA isoacceptors (including tRNATrP and tRNATYr, fQr which both modified aminoacyl-tRNAs were accessible by enzymatic aminoacylation) were used in gel filtration experiments to permit direct measurement of the individual aminoacyl-tRNA-EF-Tu-GTP complexes. These experiments were also done in the presence of equimolar amounts of the corresponding unmodified [14Claminoacyl-tRNAs, and the relative affinities for a limiting amount of EF-TuGTP were measured. The results were completely consistent with those obtained by the deacylation procedure and indicated that EF-Tu can bind to both positionalisomers of aminoacyl-tRNA with no

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Isoleucyl‐tRNA synthetase from baker's yeast: The 3′‐hydroxyl group of the 3′‐terminal ribose is essential for preventing misacylation of tRNA^Ile‐C‐C‐A with misactivated valine

Cited by 43 publications

References 10 publications

Isoleucyl‐tRNA synthetase from baker's yeast

Isoleucyl‐tRNA synthetase from baker's yeast

Plasticity of Recognition of the 3′-End of Mischarged tRNA by Class I Aminoacyl-tRNA Synthetases

Isomeric aminoacyl-tRNAs are both bound by elongation factor Tu.

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Isoleucyl‐tRNA synthetase from baker's yeast: The 3′‐hydroxyl group of the 3′‐terminal ribose is essential for preventing misacylation of tRNAIle‐C‐C‐A with misactivated valine

Cited by 43 publications

References 10 publications

Isoleucyl‐tRNA synthetase from baker's yeast

Isoleucyl‐tRNA synthetase from baker's yeast

Plasticity of Recognition of the 3′-End of Mischarged tRNA by Class I Aminoacyl-tRNA Synthetases

Isomeric aminoacyl-tRNAs are both bound by elongation factor Tu.

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Product

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Isoleucyl‐tRNA synthetase from baker's yeast: The 3′‐hydroxyl group of the 3′‐terminal ribose is essential for preventing misacylation of tRNA^Ile‐C‐C‐A with misactivated valine