Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study

Vörtler, C. Stefan; Fedorova, Olga; Persson, Therese; Kutzke, Ursula; Eckstein, Fritz

doi:10.1017/s1355838298980967

Cited by 33 publications

(19 citation statements)

References 40 publications

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“…In other tRNA systems, removal of the vicinal hydroxyl decreases aminoacylation by factors that range from a low of only 2-fold (16) to a moderate 35-fold (27), to a high of 100-fold (28). Loss of adjacent hydroxyl groups on A76 has also been shown to decrease tRNA-dependent editing (29).…”

Section: Involvement Of the 2-oh Of A76 In A Proton Relay For Aminoacylmentioning

confidence: 99%

RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNA ^Thr A76 promotes aminoacylation by threonyl-tRNA synthetase

Minajigi

Francklyn

2008

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

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Aminoacyl-tRNA synthetases (aaRSs) join amino acids to 1 of 2 terminal hydroxyl groups of their cognate tRNAs, thereby contributing to the overall fidelity of protein synthesis. In class II histidyltRNA synthetase (HisRS) the nonbridging Sp-oxygen of the adenylate is a potential general base for aminoacyl transfer. To test for conservation of this mechanism in other aaRSs and the role of terminal hydroxyls of tRNA in aminoacyl transfer, we investigated the class II Escherichia coli threonyl-tRNA synthetase (ThrRS). As with other class II aaRSs, the rate-determining step for ThrRS is amino acid activation. In ThrRS, however, the 2-OH of A76 of tRNA Thr and a conserved active-site histidine (His-309) collaborate to catalyze aminoacyl transfer by a mechanism distinct from HisRS. Conserved residues in the ThrRS active site were replaced with alanine, and then the resulting mutant proteins were analyzed by steady-state and rapid kinetics. Nearly all mutants preferentially affected the amino acid activation step, with only a modest effect on aminoacyl transfer. By contrast, H309A ThrRS decreased transfer 242-fold and imposed a kinetic block to CCA accommodation. His-309 hydrogen bonds to the 2-OH of A76, and substitution of the latter by hydrogen or fluorine decreased aminoacyl transfer by 763-and 94-fold, respectively. The proton relay mechanism suggested by these data to promote aminoacylation is reminiscent of the NAD ؉ -dependent mechanisms of alcohol dehydrogenases and sirtuins and the RNA-mediated catalysis of the ribosomal peptidyl transferase center.proton relay ͉ threonine ͉ translation ͉ aminoacyl-tRNA synthetase ͉ transient kinetics T he attachment of specific amino acids to their cognate tRNAs by aminoacyl tRNA synthetases (aaRSs) enables ribosomes to assemble amino acids into proteins accurately, as dictated by the sequence of codons in the mRNA. Two distinct classes of aaRSs catalyze aminoacylation, which occurs in 2 steps. During amino acid activation, the cognate amino acid is condensed with ATP to form an enzyme-bound adenylate, with release of pyrophosphate. This is followed by aminoacyl transfer, where the amino acid is transferred to either the 2Ј (class I aaRSs) or 3Ј (class II aaRSs) hydroxyl of A76 to generate aminoacylated tRNA, along with AMP (1, 2).The active sites of aaRSs from both classes are remarkably devoid of candidate residues to serve as general bases for aminoacyl transfer. On the basis of structural information in the GlnRS system (3, 4) and later rapid kinetics studies using histidyl-tRNA synthetase (HisRS), the pro-S nonbridging oxygen of the adenylate was proposed to serve as a general base for aminoacyl transfer (5). The latter study also highlighted a role for tRNA in modulating amino acid activation, marking it as the putative rate-determining step for overall aminoacylation. The generality of these proposals has not yet been investigated in detail, even among aaRSs in the same class.Among class II aaRSs, threonyl-tRNA synthetase (ThrRS) shares the canonical class IIa catalyt...

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Section: Involvement Of the 2-oh Of A76 In A Proton Relay For Aminoacylmentioning

confidence: 99%

RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNA ^Thr A76 promotes aminoacylation by threonyl-tRNA synthetase

Minajigi

Francklyn

2008

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

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“…These interactions facilitate transfer of the amino acid to the 3'-OH and position the nucleophile hydroxyl for attack (26). Replacing the 2'-OH with a proton results in 25-fold reduction of catalytic efficiency (27); mutation of hydrogen bonding partners results in 25−100 fold reduction of efficiency, with minimal effect on tRNA binding (28). Kinetic studies on charging of other dA76-substituted tRNAs show the range of the vicinal hydroxyls' importance for different synthetases.…”

Section: Aminoacylation Of Trnasmentioning

confidence: 99%

Participation of the tRNA A76 Hydroxyl Groups throughout Translation

Weinger

Strobel

2006

Biochemistry

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The free 2'-3' cis-diol at the 3'-terminus of tRNA provides a unique juxtaposition of functional groups that play critical roles during protein synthesis. The translation process involves universally conserved chemistry at almost every stage of this multistep procedure, and the 2'- and 3'-OHs are in the immediate vicinity of chemistry at each step. The cis-diol contribution affects steps ranging from tRNA aminoacylation to peptide bond formation. The contributions have been studied in assays related to translation over a period that spans at least three decades. In this review, we follow the 2'- and 3'-OHs through the steps of translation and examine the involvement of these critical functional groups.

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“…At the single site (d34) where substitution has no effect on binding, no hydrogen bond is observed. The magnitudes of the increase in k off upon substitution of a single ribose are generally in the range seen for the same substitution in other RNA-protein complexes, [18][19][20][21][22] although the large nearly 70-fold effect seen for the tRNA with 2 0 -deoxy modification at position 36 is only observed infrequently. While it is always difficult to rationalize the magnitude of a thermodynamic effect in terms of a structure, it is particularly difficult in this case because abundant structural and kinetic data indicate that a largescale ribosomal conformational change occurs upon tRNA binding to the ribosome.…”

mentioning

confidence: 92%

Quantitative Analysis of Deoxynucleotide Substitutions in the Codon–Anticodon Helix

Fahlman

Olejniczak

Uhlenbeck

2006

Journal of Molecular Biology

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Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study

Cited by 33 publications

References 40 publications

RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNA ^Thr A76 promotes aminoacylation by threonyl-tRNA synthetase

RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNA ^Thr A76 promotes aminoacylation by threonyl-tRNA synthetase

Participation of the tRNA A76 Hydroxyl Groups throughout Translation

Quantitative Analysis of Deoxynucleotide Substitutions in the Codon–Anticodon Helix

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Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study

Cited by 33 publications

References 40 publications

RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNA Thr A76 promotes aminoacylation by threonyl-tRNA synthetase

RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNA Thr A76 promotes aminoacylation by threonyl-tRNA synthetase

Participation of the tRNA A76 Hydroxyl Groups throughout Translation

Quantitative Analysis of Deoxynucleotide Substitutions in the Codon–Anticodon Helix

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RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNA ^Thr A76 promotes aminoacylation by threonyl-tRNA synthetase

RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNA ^Thr A76 promotes aminoacylation by threonyl-tRNA synthetase