The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae.
The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro. We show, using the two-hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo. The ability of Sup45p C-terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro. Although overexpression of either the SUP45 or SUP35 genes alone did not reduce the efficiency of codon-specific tRNA nonsense suppression, the simultaneous overexpression of both the SUP35 and SUP45 genes in nonsense suppressor tRNA-containing strains produced an antisuppressor phenotype. These data are consistent with Sup35p and Sup45p forming a complex with release factor properties. Furthermore, overexpression of either Xenopus or human eRF1 (SUP45) genes also resulted in anti-suppression only if that strain was also overexpressing the yeast SUP35 gene. Antisuppression is a characteristic phenotype associated with overexpression of both prokaryote and mitochondrial release factors. We propose that Sup45p and Sup35p interact to form a release factor complex in yeast and that Sup35p, which has GTP binding sequence motifs in its C-terminal domain, provides the GTP hydrolytic activity which is a demonstrated requirement of the eukaryote translation termination reaction.
Stansfield
I
I
Research School of Biosciences, University of Kent, Canterbury, UK.
Jones
K M
KM
Kushnirov
V V
VV
Dagkesamanskaya
A R
AR
Poznyakovski
A I
AI
Paushkin
S V
SV
Nierras
C R
CR
Cox
B S
BS
Ter-Avanesyan
M D
MD
Tuite
M F
MF
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005800:Genes, Fungal; D006639:Histidine; D008969:Molecular Sequence Data; D005810:Multigene Family; D009838:Oligodeoxyribonucleotides; D010443:Peptide Chain Termination, Translational / Q000235:genetics*; D010454:Peptide Termination Factors; D010957:Plasmids; D011328:Prions; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013489:Suppression, Genetic
D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D010454:Peptide Termination Factors; D011328:Prions; D011994:Recombinant Proteins; C068068:SUP35 protein, S cerevisiae; C080775:SUP45 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine