Abstract
Termination of translation in eukaryotes is triggered by two polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3. eRF1 is a three-domain protein that interacts with eukaryotic class 2 polypeptide chain release factor 3 via its C-terminal domain (C-domain). The high-resolution NMR structure of the human C-domain (residues 277–437) has been determined in solution. The overall fold and the structure of the β-strand core of the protein in solution are similar to those found in the crystal structure. The structure of the minidomain (residues 329–372), which was ill-defined in the crystal structure, has been determined in solution. The protein backbone dynamics, studied using 15N-relaxation experiments, showed that the C-terminal tail 414–437 and the minidomain are the most flexible parts of the human C-domain. The minidomain exists in solution in two conformational states, slowly interconverting on the NMR timescale. Superposition of this NMR solution structure of the human C-domain onto the available crystal structure of full-length human eRF1 shows that the minidomain is close to the stop codon-recognizing N-terminal domain. Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor. The results provide new insights into the possible role of the C-domain in the process of translation termination.
Highlights
Termination of translation in eukaryotes is governed by the cooperative action of two interacting polypeptide chain factors, eukaryotic class 1 polypeptide chain release factor and eukaryotic class 2 polypeptide chain release factor 3
The major functions of eukaryotic class 1 polypeptide chain release factor (eRF1) include recognition of each of the three stop codons (UAA, UAG, or UGA) in the decoding center of the small ribosomal subunit and the subsequent peptidyl-tRNA hydrolysis. eRF3 is a ribosome-dependent and eRF1-dependent GTPase encoded by an essential gene that enhances the termination efficiency by stimulating the activity of eRF1 [1,2,3,4]
For several residues situated between positions 329 and 372 a duplicated set of signals of approximately equal intensity was observed (Fig. 1; Fig. S1). This clearly indicates the presence of two conformational states of residues 329–372 of eRF1, which is highly enriched in polar and charged residues
Summary
Termination of translation in eukaryotes is governed by the cooperative action of two interacting polypeptide chain factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3 (eRF3). As residues 300–303 and 411–412 correspond to the b-sheets in the central hydrophobic core of the C-domain, it might be expected that truncation of these residues would lead to destabilization of the whole structure. This suggestion is in full agreement with recent studies on the yeast Y410S C-domain mutant [18]. In the currently available crystal structure of human eRF1 [19], coordinates exist only for the atoms that belong to the main rigid core of the C-domain, and the. More recently [20], the crystal structure of human eRF1 in a complex with the truncated form of eRF3 (residues 467–662) has been solved. On the basis of the structural data, we have performed a mutational analysis of the C-domain and investigated the impact of the mutants on stop codon recognition
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