Mutation at tyrosine in AMLRY (GILRY like) motif of yeast eRF1 on nonsense codons suppression and binding affinity to eRF3

Abstract

Termination translation in Saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. Two regions in human eRF1, position at 281-305 and position at 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed and characterized yeast eRF1 mutant at position 410 (correspond to 415 human eRF1) from tyrosine to serine residue resulting eRF1(Y410S). The mutations did not affect the viability and temperature sensitivity of the cell. The stop codons suppression of the mutant was analyzed in vivo using PGK-stop codon-LACZ gene fusion and showed that the suppression of the mutant was significantly increased in all of codon terminations. The suppression on UAG codon was the highest increased among the stop codons by comparing the suppression of the wild type respectively. In vitro interaction between eRF1 (mutant and wild type) to eRF3 were carried out using eRF1-(His)6 and eRF1(Y410S)-(His)6 expressed in Escherichia coli and indigenous Saccharomyces cerevisiae eRF3. The results showed that the binding affinity of eRF1(Y410S) to eRF3 was decreased up to 20% of the wild type binding affinity. Computer modeling analysis using Swiss-Prot and Amber version 9.0 programs revealed that the overall structure of eRF1(Y410S) has no significant different with the wild type. However, substitution of tyrosine to serine triggered the structural change on the other motif of C-terminal domain of eRF1. The data suggested that increasing stop codon suppression and decreasing of the binding affinity of eRF1(Y410S) were probably due to the slight modification on the structure of the C-terminal domain.

Keywords: Saccharomyces cerevisiae; binding affinity; eRF1; eRF3; nonsense codon suppression; termination translation.

Figure 1

Shuffling strategy and the phenotype of the transformants. A. Strategy used for plasmid shuffling. (1), LEU2 based plasmid was introduced to ∆LE2(803). (2), double transformant was iduced on media containing 5-FOA. B. Phenotypes of transformants. ∆LE2(SUP45) and ∆LE2(Y410S) grown on rich medium (Y8), SM medium without leucine (-Leu), SM medium without uracil (-Ura).

Figure 1

Shuffling strategy and the phenotype of the transformants. A. Strategy used for plasmid shuffling. (1), LEU2 based plasmid was introduced to ∆LE2(803). (2), double transformant was iduced on media containing 5-FOA. B. Phenotypes of transformants. ∆LE2(SUP45) and ∆LE2(Y410S) grown on rich medium (Y8), SM medium without leucine (-Leu), SM medium without uracil (-Ura).

Figure 2

β- galactosidase assay for ∆LE2(SUP45) and ∆LE2(Y410S). Vertical axis for % of suppression compared to β- galactosidase activity transformant carrying plasmid pUKC815.

Figure 3

SDS-PAGE electrophoregram of crude extract of E. coli overexpressed eRF1-(His)6 and eRF1(Y410S)-(His)6.; It is in first without IPTG induction and then with IPTG induction for both couple of lanes 2-3 and 4-5; lanes 1 and 6 are protein molecular markers.

Figure 4

SDS-PAGE electrophoregram of eRF3 bound to eRF1(His)6 resin and densitometric quantitation. A. Electrophoregram of eRF3 bound to eRF1(His)6 resin. Lane 1 and 6, purified eRF3 carrying His tag overexpressed in yeast. Lane 2 and 3, eRF1-Y410S(His)6 resins with (sample) and without (control) eRF3 bound. Lane 4 and 5, eRF1(His)6 resins without (control) and with (sample) eRF3 bound. B. Comparison between the ratio of eRF3 : eRF1(His)6 and eRF3 : eRF1-Y410S(His)6. The ratio between eRF3 : eRF1(His)6 used as control (100%).

Figure 5

Three dimensional structure of eRF1 and eRF1-Y410S modelling. A. Superposition of eRF1 (red) and eRF1-Y410S (yellow) structures; domain 3 of eRF1 (circle). B and C. Close up structure of eRF1 and eRF1-Y410 domain 3; position of mutation (1), position of structural change before (B) and after (C) mutation (2, and 3).

Figure 6

Homological of C-terminal among human, yeast and Y410S of eRF1. 1dt9A, human eRF1; s for beta sheet; h for helix; red bar showed secondary structure change; blue bar showed position of mutation.

Figure 7

Ramachandran plot for 11 amino acid residues including 7 amino acid residues changed from non-helix to helix form. A for yeast eRF1; B for eRF1(Y410S).