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

Abstract

In eukaryotes, a single translational release factor, eRF1, deciphers three stop codons, although its decoding mechanism remains puzzling. In the ciliate Tetrahymena thermophila, UAA and UAG codons are reassigned to Gln codons. A yeast eRF1-domain swap containing Tetrahymena domain 1 responded only to UGA in vitro and failed to complement a defect in yeast eRF1 in vivo at 37 degrees C. This finding demonstrates that decoding specificity of eRF1 from variant code organisms resides at domain 1. However, the wild-type eRF1 hybrid fully restored the growth of eRF1-deficient yeast at 30 degrees C. Tetrahymena eRF1 contains a variant sequence, KATNIKD, at the tip of domain 1. The TASNIKD variant of hybrid eRF1 rendered the eRF1-nullified yeast viable, although in an in vitro assay, the same hybrid eRF1 responded only to UGA. Nevertheless, the yeast eRF1 bearing the KATNIKD motif instead of the TASNIKS heptapeptide present in higher eukaryotes remains omnipotent in vivo. Collectively, these data suggest that variant genetic code organisms like Tetrahymena have an intrinsic potential to decode three stop codons in vivo, and that interaction within domain 1 between the KAT tripeptide and other sequences modulates the decoding specificity of Tetrahymena eRF1.

Figure 1

Strategy of domain swapping between Sp-eRF1 and Tt-eRF1. (A) The three-dimensional structure of human eRF1 (PDB ID code 1DT9). Each domain was swapped at the hinge regions indicated by arrows. The TASNIKS heptapeptide and the GGQ tripeptide are shown at the tips of domains 1 and 2, respectively. (B) Conserved amino acid sequences at the junctions of domains 1–2 and 2–3 of eRF1s. Amino acids identical to those of Tetrahymena eRF1 are shown by outlined characters; restriction enzyme sites used for domain swapping are shown by triangles. Species abbreviations: MT, Methanobacterium thermoautotrophicum; HS, Homo sapiens; CE, S. cerevisiae; SP, S. pombe; and TT, T. thermophila.

Figure 2

In vivo complementation test of the temperature-sensitive eRF1 (sup45 ts) strain of S. cerevisiae by wild-type and variant hybrid eRF1s. (A) Amino acid sequence comparison of the TASNIKS heptapeptide (boxed) and surrounding regions of eRF1s from human, yeast, and ciliates. Residues identical and similar to those in S. pombe eRF1 are shown by outlined characters and gray boxes, respectively. (B) Growth of transformants at permissive (30°C) and nonpermissive (37°C) temperatures. MT557/1d (sup45 ts) cells were transformed with pYX112 derivatives encoding Sp-eRF1 and wild-type or variant ΨeRF1 proteins. Ura⁺ transformants were selected at 30°C, and their growth was monitored at 37°C. Amino acid changes introduced into the wild-type (KATNIKD) heptapeptide of ΨeRF1 are shown by outlined characters.

Figure 3

The capacity of wild-type and variant ΨeRF1 proteins to complement the nullified eRF1 gene of S. cerevisiae. (A) The gene disruption of S. cerevisiae eRF1. The LUE2 marker was inserted into the HpaI-EcoRV sites of the SUP45 sequence cloned in plasmid pET-Sc-eRF1. Numbers refer to the initiator codon of the eRF1 gene. The DNA containing the nullified Δsup45∷LEU2 allele was amplified by PCR using multicloning site primers that flank the sup45 insert and transformed into MT557/1d (sup45 ts) cells in the presence of pYX112 plasmids encoding Sp-eRF1 and wild-type (KATNIKD) or variant (TASKINS and TASNIKD) ΨeRF1s; Leu⁺ (Ura⁺) transformants were selected, and those whose chromosomal copy of the eRF1 (sup45 ts) sequence was replaced by the Δsup45∷LEU2 allele were isolated. (B) DNA analyses of the disruption of chromosomal copy of eRF1 in S. cerevisiae transformants. The DNAs containing the insert were amplified from Leu⁺ transformants obtained in A by PCR using primers 5′-TATTGAGATCTGGAAGGTCAAGAAGTTGG-3′ and 5′-GTTGATAGGTTTGTAAGGTTCGATGTC-3′ shown by arrows in A. These two primer sequences were chosen from S. cerevisiae eRF1 and do not crossreact with Tetrahymena eRF1 or S. pombe eRF1 sequences. Samples used for PCR amplification: lane 1, plasmid DNA encoding the wild-type eRF1 of S. cerevisiae (control); lane 2, plasmid DNA encoding the Δsup45∷LEU2 eRF1 of S. cerevisiae (control); lanes 3–6, Leu⁺ transformant DNAs selected as in A, in which the chromosomal copy of eRF1 was (lanes 4–6) or was not (lane 3) disrupted by the LEU2 insert. Lanes 4, 5, and 6 represent Leu⁺ transformants expressing wild-type (KATNIKD) and variant (TASNIKS, TASNIKD) ΨeRF1 proteins, respectively. (C) The growth of the eRF1-nullified S. cerevisiae cells in the presence of plasmids encoding Sp-eRF1 and wild-type or variant ΨeRF1 proteins at 30°C and 37°C.