Different modes of stop codon restriction by the Stylonychia and Paramecium eRF1 translation termination factors.
In universal-code eukaryotes, a single-translation termination factor, eukaryote class-1 polypeptide release factor (eRF1), decodes the three stop codons: UAA, UAG, and UGA. In some ciliates, like Stylonychia and Paramecium, eRF1s exhibit UGA-only decoding specificity, whereas UAG and UAA are reassigned as sense codons. Because variant-code ciliates may have evolved from universal-code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of the protein. Using both in vitro and in vivo assays, we show here that chimeric molecules composed of the N-terminal domain of Stylonychia eRF1 fused to the core domain (MC domain) of human eRF1 retained specificity toward UGA; this unambiguously associates eRF1 stop codon specificity to the nature of its N-terminal domain. Functional analysis of eRF1 chimeras constructed by swapping ciliate N-terminal domain sequences with the matching ones from the human protein highlighted the crucial role of the tripeptide QFM in restricting Stylonychia eRF1 specificity toward UGA. Using the site-directed mutagenesis, we show that Paramecium eRF1 specificity toward UGA resides within the NIKS (amino acids 61-64) and YxCxxxF (amino acids 124-131) motifs. Thus, we establish that eRF1 from two different ciliates relies on different molecular mechanisms to achieve specificity toward the UGA stop codon. This finding suggests that eRF1 restriction of specificity to only UGA might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UAG and UAA to sense codons.
Lekomtsev
Sergey
S
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia.
Kolosov
Petr
P
Bidou
Laure
L
Frolova
Ludmila
L
Rousset
Jean-Pierre
JP
Kisselev
Lev
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016798:Ciliophora / Q000235:genetics*; D018388:Codon, Terminator; D006801:Humans; D010247:Paramecium / Q000235:genetics; D010454:Peptide Termination Factors / Q000235:genetics* / Q000502:physiology; D014176:Protein Biosynthesis / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins; D013379:Substrate Specificity
D018388:Codon, Terminator; D010454:Peptide Termination Factors; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins