Mutation at tyrosine in AMLRY (GILRY like) motif of yeast eRF1 on nonsense codons suppression and binding affinity to eRF3.
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.
Akhmaloka
Biochemistry Research Division, Faculty of Mathematics, Natural Sciences, Institut Teknologi Bandung, Jln Ganesha 10, Bandung, Indonesia. loka@chem.itb.ac.id
Susilowati
Prima Endang
PE
Subandi
Madayanti
Fida
F
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D018389:Codon, Nonsense; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008956:Models, Chemical; D008958:Models, Molecular; D010454:Peptide Termination Factors / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry*
D018389:Codon, Nonsense; D010454:Peptide Termination Factors; C080775:SUP45 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C087971:peptide-chain-release factor 3; D014443:Tyrosine