Fig. 1. Cross-linking patterns obtained with 42mer mRNA analogs containing stop codons (UAG, UAA or UAG) or a sense codon (UCA) in presence of C-terminally His-tagged human wild-type eRF1 (wt) or mutated human eRF1 containing either S64D or I35V-L126F substitutions. After irradiation, the reaction products were separated onto a 10% SDS–polyacrylamide gel and analyzed by autoradiography. A control reaction without eRF1 (No eRF1) is shown for each mRNA analog. The 68 kDa band corresponding to the eRF1–mRNA cross-link is indicated by an arrow. Molecular mass markers in kDa are indicated on the left.

Fig. 3. Comparison of the cross-linking pattern of human eRF1 (Hs-eRF1) with recombinant Eu-eRF1(1–224). Eu-eRF1(1–224) contains residues 1–224 from E.aediculatus eRF1 and residues 225–435 from human eRF1. (A) Schematic representation of the amino acid sequences of Hs-eRF1 and recombinant Eu-eRF1(1–224). The approximate locations of the NIKS (domain 1) and GGQ (domain 2) motifs are indicated. The region of Euplotes eRF1 in Eu-eRF1(1–224) is shaded in light gray. (B) Cross-linking patterns of 42mer mRNA analogs containing UGA, UAA, UAG, UCA or UGG codons (as indicated below the autoradiogram) in the presence of Hs-eRF1 or Eu-eRF1(1–224) as indicated above the autoradiograms. The cross-linking pattern of the UGA mRNA analog in the absence of eRF1 is shown in lane 0. The irradiated reactions were analyzed by 7.5% SDS–PAGE. The region containing the eRF1–mRNA cross-links is boxed with broken line. (C) Enlargement views of regions boxed with broken lines in (B). Cross-links between mRNA analogs containing the canonical stop (UGA, UAA, UAG) or sense (UGG and UCA) codons as indicated below the autoradiograms and Hs-eRF1 (upper panel) or recombinant Eu-eRF1(1–224) (lower panel). The cross-linking pattern of the UGA mRNA analog in the absence of eRF1 is shown in lane 0. An asterisk indicates a Hs-eRF1–mRNA cross-link and a hash symbol indicates a Eu-eRF1(1–224)–mRNA cross-link.

Fig. 5. The structure of eRF1. (A) Crystal structure of human eRF1 with the ribbon representation of the secondary structure. Major domains and secondary structure elements are labeled. Functionally important motifs are indicated by an arrow with the one letter amino acid code (GGQ and NIKS). In domain 1, the different colors indicate the regions of human eRF1 substituted by regions from Euplotes eRF1: amino acids 1–34 are colored pink, amino acids 35–51 are in dark blue, amino acids 52–68 are in green, amino acids 69–94 are in yellow and amino acids 95–145 are in red. Domain 2 is colored light blue and domain 3 is in gray. The coordinate data were obtained from the Protein Data Bank (accession code ccss 1TD9). (B) Enlargement of domain 1. (C) Linear representation of domain 1 (residues 1–145). The regions of human eRF1 swapped for Euplotes eRF1 are represented in cylinders (α-helices) and large arrows (β-strands) using the same colors as in (A). The positions of the junctions (35, 52, 68, 94) are indicated.

Fig. 2. Analysis of the cross-linking patterns obtained in the presence of recombinant human eRF1 containing region 52–68 from either E.aediculatus, Eu-eRF1(52–68) or T.thermophila, Tt-eRF1(52–68). (A) Comparison of eRF1 amino acid sequences from Human (Hs, DDBJ/EMBL/GenBank accession No. P46055), E.aediculatus (Eu, accession No. AAK07830) and T.thermophila (Tt, accession No. BAA85336). The alignment is shown only for the positions 40–71. The swapped region between Euplotes, Tetrahymena and human eRF1, residues 52–68, is boxed. Identical amino acids residues are shaded in black. (B) Cross-linking patterns with 42mer mRNA analogs containing UGA, UAA, UAG, UCA codons (indicated below the autoradiogram) in the presence of recombinant eRF1, Eu-eRF1(52–68) or Tt-eRF1(52–68) as indicated above. The cross-linking pattern of the UGA mRNA analog in the absence of eRF1 is shown in lane 0. The irradiated reactions were separated on a 7.5% SDS–polyacrylamide gel. eRF1–mRNA cross-links are indicated by an arrow.

Fig. 4. Localization of the Euplotes eRF1 region implicated in stop codon discrimination. (A) Schematic representation of the human– Euplotes hybrid eRF1s constructed. The regions of Euplotes eRF1 are shaded in light gray. The approximate locations of the NIKS and GGQ motifs are indicated. Numbering is according to human eRF1 amino acids sequence. (B) mRNA–eRF1 cross-links obtained for the recombinant eRF1s are indicated on the left. The 42mer mRNA analogs containing the canonical stop (UGA, UAA, UAG) or sense (UAC, UGG) codons are indicated. The irradiated reactions were analyzed by 7.5% SDS–PAGE. Only the mRNA–eRF1 cross-linking regions of the autoradiograms are shown (as in Figure 3C), and the eRF1–mRNA cross-links are marked by an arrow.

Fig. 6. Schematic representation of plasmid pET21b derivatives expressing C-terminally His-tagged eRF1 from human (pET-Hs-eRF1-His₆) or Euplotes (pET-Eu-eRF1-His₆) under the control of the T7 promoter (T7p). The restrictions sites used for the construction of the recombinant eRF1s are indicated. The restriction sites indicated under pET-Hs-eRF1-His₆ were introduced by site-directed mutagenesis.