Construction of heterodimer tyrosyl-tRNA synthetase shows tRNATyr interacts with both subunits.

Carter, Paul; Bedouelle, Hugues; Winter, Greg

doi:10.1073/pnas.83.5.1189

Cited by 63 publications

(48 citation statements)

References 31 publications

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“…Apart from a very few exceptions (50), the oligomeric structure of aminoacyl-tRNA synthetases specific for particular amino acids is conserved in evolution while this does not seem to be the case for the number of tRNAs simultaneously bound per dimer. The most studied tyrosyl-tRNA synthetase isolated from Bacillus stearothermophilus binds one tRNA across two subunits (51). A similar type of complex was detected between Thermus thermophilus SerRS and its cognate tRNA (46) while two molecules of tRNA Ser interact with dimeric SerRS from Escherichia coli (46,52).…”

Section: What Is the Fate Of Noncovalent Complexes During Maldimentioning

confidence: 82%

Detection of Noncovalent tRNA·Aminoacyl-tRNA Synthetase Complexes by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Gruić-Sovulj¹,

Lüdemann²,

Hillenkamp³

et al. 1997

Journal of Biological Chemistry

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Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-MS) was used for the study of complexes formed by yeast seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) with tRNA Ser and tRNA Tyr . Cognate and noncognate complexes were easily distinguished due to a large mass difference between the two tRNAs. Both homodimeric synthetases gave MS spectra indicating intact desorption of dimers. The spectra of synthetase-cognate tRNA mixtures showed peaks of free components and peaks assigned to complexes. Noncognate complexes were also detected. In competition experiments, where both tRNA species were mixed with each enzyme only cognate ␣ 2 ⅐tRNA complexes were observed. Only cognate ␣ 2 ⅐tRNA 2 complexes were detected with each enzyme. These results demonstrate that MALDI-MS can be used successfully for accurate mass and, thus, stoichiometry determination of specific high molecular weight noncovalent protein-nucleic acid complexes.

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Section: What Is the Fate Of Noncovalent Complexes During Maldimentioning

confidence: 82%

Detection of Noncovalent tRNA·Aminoacyl-tRNA Synthetase Complexes by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Gruić-Sovulj¹,

Lüdemann²,

Hillenkamp³

et al. 1997

Journal of Biological Chemistry

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“…T51P, I52L and I52L-L105V had no detectable effect on the global thermodynamic stability of TyrRS(Á1) but they decreased the stability of the association between its subunits. As the monomer of TyrRS(Á1) is inactive, a lower stability of the association between the subunits implied a lower functional stability Carter et al, 1986). Thus, three residues of the dense cluster under study, Thr51, Ile52 and Leu105, were involved in the stability of TyrRS(Á1).…”

Section: Discussionmentioning

confidence: 99%

Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization 1 1Edited by A. R. Fersht

Park

Guez

Bedouelle

1999

Journal of Molecular Biology

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A dense cluster of eight residues was identi®ed at the crossing of two a-helices in tyrosyl-tRNA synthetase (TyrRS) from the thermophile Bacillus stearothermophilus. Its mechanism of evolution was characterized. Four residues of this cluster are not conserved in TyrRS from the mesophile Escherichia coli. The corresponding mutations were constructed in TyrRS(Á1), a derivative of TyrRS from B. stearothermophilus in which the anticodon binding domain is deleted. Mutations I52L (i.e. Ile52 into Leu), M55L and L105V did not affect the activity of TyrRS(Á1) in the pyrophosphate exchange reaction whereas T51P increased it. The kinetic stabilities of TyrRS(Á1) and its mutant derivatives at 68.5 C were determined from experiments of irreversible thermal precipitation. They were in the order L105V < I52L < T51P < Wild Type 4 M55L; mutation I52L partially compensated L105V in these experiments whereas M55L was coupled neither to I52L nor to L105V. Mutations I52L and L105V affected the stability of the dimeric TyrRS(Á1) at different steps of its unfolding by urea, monitored under equilibrium conditions by spectro¯uorometry or size exclusion chromatography. I52L destabilized the association between the subunits even though residue Ile52 is more than 20 A Ê away from the subunit interface. L105V destabilized the monomeric intermediate of unfolding. The two mutational pathways, going from the wildtype TyrRS(Á1) to the I52L-L105V double mutant through each of the single mutants were not equivalent for the stability of the monomeric intermediate and for the total stability of the dimer. One pathway contained two neutral steps whereas the other pathway contained a destabilizing step followed by a stabilizing step. Mutation I52L allowed L105V along the ®rst pathway and compensated it along the second pathway. Thus, the effects of I52L and L105V on stability depended on the structural context. The gain in activity due to T51P was at the expense of a slight destabilization.

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“…Nonetheless, some elegant experiments have been carried out to delineate the likely surface residues of the crystallographically disordered C-terminal segment of the polypeptide (Bedouelle & Winter, 1986;Carter et al, 1986). By a combined use of a deletion mutant lacking the tRNA binding domain and point mutations at the activation site it has been possible to show, through the use of hybrid dimers prepared in vitro, that tRNA probably binds to both subunits of the dimer.…”

Section: Al-antitrypsinmentioning

confidence: 99%

Protein engineering. The design, synthesis and characterization of factitious proteins

Shaw

1987

Biochemical Journal

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Construction of heterodimer tyrosyl-tRNA synthetase shows tRNATyr interacts with both subunits.

Abstract: The tyrosyl-tRNA synthetase (EC 6.1

Cited by 63 publications

References 31 publications

Detection of Noncovalent tRNA·Aminoacyl-tRNA Synthetase Complexes by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Detection of Noncovalent tRNA·Aminoacyl-tRNA Synthetase Complexes by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization 1 1Edited by A. R. Fersht

Protein engineering. The design, synthesis and characterization of factitious proteins

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