Ribosomal association of the yeast SAL4 (SUP45) gene product: implications for its role in translation fidelity and termination

The SAL4 gene of the yeast Saccharomyces cerevisiae encodes a novel translation factor (Sal4p) involved in maintaining translational fidelity. Using a polyclonal antibody raised against a Sal4p-beta-galactosidase fusion protein, Sal4p was shown to be almost exclusively associated with the ribosomal fraction. Even when the ribosomes were treated with 0.8 M KCl, only low levels of Sal4p were detected in the post-ribosomal supernatant, suggesting a very strong affinity between Sal4p and the ribosome. Analysis of the distribution of Sal4p in the ribosomal population revealed that it was principally associated with 40S subunits, monosomes and polysomes. Incubation in high salt concentrations (0.8 M KCl) suggested that the affinity of Sal4p for the 40S subunit was lower than that for monosomes or polysomes. The Sal4p:ribosome association was only maintained when ribosomes were prepared in the presence of the translation elongation inhibitor cycloheximide; in uninhibited cells much lower levels of Sal4p were detectable in the 'run-off' polysomes. In view of these data, and given the stoichiometry of Sal4p to individual ribosomal proteins (estimated at less than 1:20), we suggest that Sal4p plays an ancillary role in translation termination.     

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.     

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum

Extremophiles - Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5’-phosphate-dependent enzymes and engaged in glycine biosynthesis from...     

Depletion in the levels of the release factor eRF1 causes a reduction in the efficiency of translation termination in yeast

In Saccharomyces cerevisiae, translation termination is mediated by a complex of two proteins, eRF1 and eRF3, encoded by the SUP45and SUP35 genes, respectively. Mutations in the SUP45 gene were selected which enhanced suppression by the weak ochre (UAA) suppressor tRNA^SerSUQ5. In each of four such allo-suppressor alleles examined, an in-frame ochre (TAA) mutation was present in the SUP45 coding region; therefore each allele encoded both a truncated eRF1 protein and a full-length eRF1 polypeptide containing a serine missense substitution at the premature UAA codon. The full-length eRF1 generated by UAA read-through was present at sub-wild-type levels. In an suq5⁺ (i.e. non-suppressor) background none of the truncated eRF1 polypeptides were able to support cell viability, with the loss of only 27 amino acids from the C-terminus being lethal. The reduced eRF1 levels in these sup45 mutants did not lead to a proportional reduction in the levels of ribosome-bound eRF3, indicating that eRF3 can bind the ribosome independently of eRF1. A serine codon inserted in place of the premature stop codon at codon 46 in the sup45–22 allele did not generate an allosuppressor pheno-type, thereby ruling out this‘missense’mutation as the cause of the allosuppressor phenotype. These data indicate that the cellular levels of eRF1 are important for ensuring efficient translation termination in yeast.     

Molecular characterization of transesterification activity of novel lipase family I.1

Lipase’s thermostability and organic solvent tolerance are two crucial properties that enable it to function as a biocatalyst. The present study examined the characteristics of two recombinant thermostable lipases (Lk2, Lk3) based on transesterification activity. Conversion of C12-C18 methyl ester with paranitrophenol was investigated in various organic solvent. Both lipases exhibited activity on difference carbon chain length (C12 - C18, C18:1, C18:2) of substrates. The activity of Lk2 was higher in each of substrate compared with that of Lk3. Experimental findings showed that the best substrates for Lk2 and Lk3 are C18:1 and C18:2 respectively, in agreement with the computational analysis. The activity of both enzymes prefers on nonpolar solvent. On nonpolar solvent the enzymes are able to keep its native folding shown by the value of radius gyration, solvent–enzyme interaction and orientation of triad catalytic residues. Lk3 appeared to be more thermostable, with maximum activity at 55°C. The presence of Fe³⁺ increased the activity of Lk2 and Lk3. However, the activity of both enzymes were dramatically decreased by the present of Ca²⁺ despite of the enzymes belong to family I.1 lipase known as calcium dependent enzyme. Molecular analysis on His loop of Lk2 and Lk3 on the present of Ca²⁺ showed that there were shifting on the orientation of catalytic triad residues. All the data suggest that Lk2 and Lk3 are novel lipase on the family I.1 and both lipase available as a biocatalyst candidate.