NMR solution structure and function of the C-terminal domain of eukaryotic class 1 polypeptide chain release factor

Abstract

Keywords human eukaryotic class 1 polypeptide chain release factor (eRF1); NMR structure and dynamics; stop codon recognition specificity; termination of protein synthesis 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 b-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 15 N-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.Abbreviations C-domain, C-terminal domain (or domain 3) of class 1 polypeptide chain release factor; DHPC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; eRF1, eukaryotic class 1 polypeptide chain release factor; eRF3, eukaryotic class 2 polypeptide chain release factor 3; HSQC, heteronuclear single quantum coherence; M-domain, eukaryotic class 1 polypeptide chain release factor middle domain (or domain 2); minidomain, residues 329-372 of human eRF1; N-domain, eukaryotic class 1 polypeptide chain release factor N-terminal domain (or domain 1); NMD, nonsense-mediated decay; PP2A, protein phosphatase 2A; RDC, residual dipolar coupling; R ex , conformational exchange contribution to R 2 ; RF, release factor; R 1 , longitudinal or spin-lattice relaxation rate; R 2 , transverse or spin-spin relaxation rate; S 2 , order parameter reflecting the amplitude of picosecond-nanosecond bond vector dynamics; s e , effective internal correlation time; s m , overall rotational correlation time.

Cryo-EM of Ribosomal 80S Complexes with Termination Factors Reveals the Translocated Cricket Paralysis Virus IRES

“…For an interpretation in molecular terms, we docked our previous cryo-EM based homology model of the human 80S ribosome (Budkevich et al, 2014), which was generated on the basis of S. cerevisiae 80S (Ben-Shem et al, 2011) and T. thermophila 40S and 60S X-ray structures (Klinge et al, 2011; Rabl et al, 2011). eRF3 was modeled based on the homology with its archaeal eEF1A ortholog (aEF1α) from the aEF1α/aRF1 complex (Kobayashi et al, 2012) and eRF1 by docking individual domains of the human factor (Mantsyzov et al, 2010; Song et al, 2000) into the cryo-EM density. The CrPV-STOP IRES has been modeled based on the previously published X-ray structure for domain 3 (Zhu et al, 2011) and cryo-EM model for domains 1 and 2 (Schüler et al, 2006).…”

“…The eS31 interaction seems to be unique for eRF1, as it could not be observed for Dom34 in the Dom34/Hbs1-complex (Becker et al, 2011) or A/T tRNA in the decoding complex (Budkevich et al, 2014) because both are lacking a similar domain. Mutational analysis indicated that the mini-domain influences the specificity of eRF1 for certain stop codons during stop-codon recognition (Mantsyzov et al, 2010), which may explain its exclusive presence in eRF1.…”

“…The initial structural model for eRF1 was built by domain-wise rigid-body fitting (Mantsyzov et al, 2010; Song et al, 2000) accompanied by MDfit (Ratje et al, 2010; Whitford et al, 2011), COOT and CNS (Brünger et al, 1998). eRF3 was modeled based on homology to aEF1a (Kelley and Sternberg, 2009; Kobayashi et al, 2012; Tung and Sanbonmatsu, 2004) and yeast eRF3 (Kong et al, 2004).…”

A genetic approach for analyzing the co-operative function of the tRNA mimicry complex, eRF1/eRF3, in translation termination on the ribosome

“…The X-ray structure of this region has been left unsolved in the structure of apo-eRF1 as well as in structures of complexes with eRF3, due to the flexibility of this domain. The NMR solution structure of domain C was solved and the extra domain, termed the ‘mini-domain’, was reported to affect codon-specificity (52). According to the latest docking model-assisted cryo-EM study (30), three mutations (E285K, D389G and G394S) identified in this study are located in this ‘mini-domain’, which interacts with the ‘40S beak’, a structural protrusion of the 40S ribosomal subunit that is close to the entry site for translation factors, while the other domain C mutations probably interact with eRF3 or the P-stalk of the large 60S subunit.…”

Structure of the mammalian ribosomal pre-termination complex associated with eRF1•eRF3•GDPNP

“…The rigid core of domain C contains a flexible insertion forming a mini-domain (8). Domain N is involved in stop codon recognition.…”