Polypeptide release factor eRF1 from Tetrahymena thermophila: cDNA cloning, purification and complex formation with yeast eRF3

Abstract

The first cDNA for the translational release factor eRF1 of ciliates was cloned from Tetrahymena thermophila. The coding frame contained one UAG and nine UAA codons that are reassigned for glutamine in Tetrahymena. The deduced protein sequence is 57% identical to human eRF1. The recombinant Tetrahymena eRF1 purified from a yeast expression system was able to bind to yeast eRF3 as do other yeast or mammalian eRF1s as a prerequisite step for protein termination. The recombinant Tetrahymena eRF1, nevertheless, failed to catalyze polypeptide termination in vitro with rat or Artemia ribosomes, at least in part, due to less efficient binding to the heterologous ribosomes. Stop codon specificity and phylogenetic significance of Tetrahymena eRF1 are discussed from the conservative protein feature.z 1999 Federation of European Biochemical Societies.

Terminating eukaryote translation: Domain 1 of release factor eRF1 functions in stop codon recognition

“…Although a direct role for the ribosome and rRNA in stop codon recognition has been proposed in the past, evidence now points to a direct recognition model for RF stop codon discrimination+ First, prokaryote RF2 can be directly UV crosslinked to the stop codon and downstream nucleotides in vitro, inferring the release factor is in intimate contact with the termination signal (Brown & Tate, 1994;Poole et al+, 1998)+ Second, overexpression of either prokaryote RF1 or eukaryote eRF1 acts to out-compete suppressor tRNA species for stop codon binding+ This so-called antisuppressor phenotype indicates both tRNAs and RFs are cognate species in direct competition for stop codon binding (Weiss et al+, 1984;Stansfield et al+, 1995a;Legoff et al+, 1997)+ Direct recognition models like these imply that RFs might act in a tRNA-like manner to discriminate between codons+ This idea was supported by the discovery that the structure of domain IV/V of elongation factor G complexed with GDP is very similar to that of a tRNA molecule when part of a ternary complex with EF-Tu and GTP, prompting the proposal that protein elongation factors might mimic tRNA molecules (Nissen et al+, 1995)+ On the basis of limited RF sequence similarity to EF-G, the concept of structural tRNA mimicry by the central domain of class I release factors was developed (Ito et al+, 1996)+ The recent solution of the crystal structure of eukaryote eRF1 has allowed a reappraisal of this model for eukaryote RFs, and it is now apparent that, although the central domain of eRF1 at least does not represent a tRNA-like structure (Song et al+, 2000), the Y-shaped eRF1 molecule does have both similar shape and overall dimensions to a tRNA+ The N-terminal domain 1 of eRF1 may represent a potential anticodonlike region, on the basis of its position relative to the peptidyl-release triggering GGQ motif (analogous to the tRNA CCA acceptor stem; Song et al+, 2000)+ Thus the eRF1-tRNA mimicry model is now supported by direct structural evidence, although it seems likely that the bacterial RFs may be structurally dissimilar to eRF1 because their predicted secondary structures are unalike+ It cannot, however, be ruled out that the bacterial RF overall shape may still mimic that of a tRNA+ Recently, the crystal structure of another ribosomal A siteinteracting protein, the bacterial ribosome recycling factor (RRF), has been solved, revealing it, too, has a tRNA-like shape, and strengthening the tRNA mimicry proposal (Selmer et al+, 1999)+ How then is stop codon recognition achieved by a tRNA-analog protein RF? In a recent study, mixed RF1/ RF2 domain hybrid proteins were constructed and screened for RF1 molecules with RF2-like stop codon specificity+ Tripeptide motifs were identified from the central D domain of both release factors that conferred codon specificity, with the first and third amino acids of this peptide discriminating the second and third purine bases of the stop codons (Ito et al+, 2000)+ These findings reinforce the proposal that bacterial RFs directly recognize the stop codon+ Eukaryote eRF1 from Tetrahymena, recently cloned (Karamyshev et al+, 1999), may exhibit natural altered stop codon recogni...…”

Omnipotent decoding potential resides in eukaryotic translation termination factor eRF1 of variant-code organisms and is modulated by the interactions of amino acid sequences within domain 1

“…An Sp-eRF1 overexpression plasmid was constructed by subcloning the NcoI-NheI segment of Sp-eRF1 into the NcoI-BamHI sites of pET15b (Novagen) by linker ligation to give rise to pET15b-Sp-eRF1. The EcoRI-Bpu1102I segments carrying the wild-type and mutant ⌿eRF1 sequences were substituted for the equivalent segment in the pET30b-based Tt-eRF1 expression plasmid, pTT-eRF1-38͞1 (29), to give rise to pET30b-⌿eRF1 and its mutant derivatives. E. coli strain BL21 (DE3) was transformed with pET15b-Sp-eRF1 and pET30b-⌿eRF1, and the transformants were grown at 37°C in 0.2 liter of LB medium containing ampicillin (100 g͞ml) until A 600 0.7 was reached.…”

“…Ciliate eRF1 genes have been cloned from T. thermophila (18), E. octacarinatus (19), E. aediculatus (17,20), and Oxytricha trifallax (UAA and UAG for Gln; ref. 20).…”

“…The authentic Tt-eRF1 is unable to catalyze polypeptide termination in vitro with mammalian ribosomes because of its inefficient binding to the heterologous ribosomes (18). Therefore, based on the structural information, domains 1-3 were swapped between Tt-eRF1 and the fission yeast Schizosaccharomyces pombe eRF1 (referred to as Sp-eRF1; ref.…”

Endless possibilities: translation termination and stop codon recognition

“…Recently, the eRF1 gene sequence from Tetrahymena thermophila has been isolated ; it encodes an eRF1 protein highly homologous at the amino acid level to other eukaryotic and archaeal release factors (e.g. 57 % identity with human eRF1), but it is unclear which, if any, amino acid substitutions are responsible for altered stop codon specificity (Karamyshev et al, 1999). However, it is known that the Tetrahymena eRF1 is unable to complement a yeast temperature-sensitive mutation in the SUP45 gene (encoding eRF1), consistent, among other possibilities, with a restricted stop codon recognition specificity (Karamyshev et al, 1999).…”