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Thermal denaturation of class 1 eukaryotic translation termination factor eRF1. Relationship between stability and functional activity of eRF1 mutants

  • Molecular Mechanisms of Biological Processes
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Abstract

Differential scanning calorimetry (DSC) was used to study thermal denaturation of the human class 1 translation termination factor eRF1 and its mutants. Free energy changes caused by amino acid substitutions in the N domain were computed for eRF1. The melting of eRF1, consisting of three domains, proved to be cooperative. The thermostability of eRF1 was not affected by certain substitutions and was slightly increased by certain others. The corresponding residues were assumed to play no role in maintaining the eRF1 structure, which agreed with the published X-ray data. In these mutants (E55D, Y125F, N61S, E55R, E55A, N61S + S64D, C127A, and S64D), a selective loss of the capability to induce hydrolysis of peptidyl-tRNA in the ribosomal P site in the presence of a stop codon was not associated with destabilization of their spatial structure. Rather, the loss was due to local changes in the stereochemistry of the side groups of the corresponding residues in functionally important sites of the N domain. Two amino acid residues of the N domain, N129 and F131, proved to play an important role in the structural stability of eRF1 and to affect the selective recognition of mRNA stop codons in the ribosome. The recognition of the UAG and UAA stop codons in vitro was more tightly associated with the stability of the spatial structure of eRF1 as compared with that of the UGA stop codon.

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Authors and Affiliations

  1. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia

    V. A. Mitkevich, A. V. Kononenko, N. J. Oparina, P. M. Kolosov, A. A. Makarov & L. L. Kisselev

  2. Center for Medical Studies, University of Oslo, Moscow, 119334, Russia

    V. A. Mitkevich

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  1. V. A. Mitkevich
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  2. A. V. Kononenko
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  3. N. J. Oparina
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  4. P. M. Kolosov
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  5. A. A. Makarov
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  6. L. L. Kisselev
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Original Russian Text © V.A. Mitkevich, A.V. Kononenko, N.J. Oparina, P.M. Kolosov, A.A. Makarov, L.L. Kisselev, 2006, published in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 1, pp. 100–110.

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Mitkevich, V.A., Kononenko, A.V., Oparina, N.J. et al. Thermal denaturation of class 1 eukaryotic translation termination factor eRF1. Relationship between stability and functional activity of eRF1 mutants. Mol Biol 40, 86–95 (2006). https://doi.org/10.1134/S0026893306010134

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  • DOI: https://doi.org/10.1134/S0026893306010134

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