Eukaryotic class 1 translation termination factor eRF1 − the NMR structure and dynamics of the middle domain involved in triggering ribosome‐dependent peptidyl‐tRNA hydrolysis

Abstract

Termination of translation, one of the most complex stages in protein biosynthesis, is regulated by the cooperative action of two interacting polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3 (eRF3). The roles of these The eukaryotic class 1 polypeptide chain release factor is a three-domain protein involved in the termination of translation, the final stage of polypeptide biosynthesis. In attempts to understand the roles of the middle domain of the eukaryotic class 1 polypeptide chain release factor in the transduction of the termination signal from the small to the large ribosomal subunit and in peptidyl-tRNA hydrolysis, its high-resolution NMR structure has been obtained. The overall fold and the structure of the b-strand core of the protein in solution are similar to those found in the crystal. However, the orientation of the functionally critical GGQ loop and neighboring a-helices has genuine and noticeable differences in solution and in the crystal. Backbone amide protons of most of the residues in the GGQ loop undergo fast exchange with water. However, in the AGQ mutant, where functional activity is abolished, a significant reduction in the exchange rate of the amide protons has been observed without a noticeable change in the loop conformation, providing evidence for the GGQ loop interaction with water molecule(s) that may serve as a substrate for the hydrolytic cleavage of the peptidyl-tRNA in the ribosome. The protein backbone dynamics, studied using 15 N relaxation experiments, showed that the GGQ loop is the most flexible part of the middle domain. The conformational flexibility of the GGQ and 215-223 loops, which are situated at opposite ends of the longest a-helix, could be a determinant of the functional activity of the eukaryotic class 1 polypeptide chain release factor, with that helix acting as the trigger to transmit the signals from one loop to the other. Abbreviations aRF1s, archaeal RFs; eRF1, eukaryotic class 1 polypeptide chain release factor; eRF3, eukaryotic class 2 polypeptide chain release factor 3; HNCA, three-dimensional experiment correlating amide HN and Ca signals; HSQC, heteronuclear single quantum coherence spectroscopy; M-domain, eRF1 middle domain (or domain 2); PTC, peptidyl transferase center of the ribosome; R 1 , longitudinal or spin-lattice relaxation rate; R 2 , transverse or spin-spin relaxation rate; R ex , conformational exchange contribution to R 2 ; RF, polypeptide chain release factor(s); S 2 , order parameter reflecting the amplitude of ps-ns bond vector dynamics; s e , effective internal correlation time; s m , overall rotational correlation time.

Sense from nonsense: therapies for premature stop codon diseases

“…eRF1 recognizes all three stop codons through an extended area in its N-terminal domain [21][22][23], although it has recently become clear that eRF1 acts differently depending on the type of codon; its behavior with UGA being different from that with UAA or UAG [21,24]. When a stop codon is recognized, the highly conserved Gly-Gly-Gln (GGQ) motif in the central domain of eRF1 triggers peptidyl-tRNA hydrolysis by activating the peptidyl transferase center of the ribosome [25][26][27]. The Cterminal domain of eRF1 is involved in binding eRF3, which is essential for in vivo termination (Figure 2a) [28].…”

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

“…The N-terminal domain (N-domain) is involved in the recognition of the stop codon [1,5,6]. The middle domain (M-domain) catalyzes the hydrolysis of the peptidyltRNA ester bond within the peptidyltransferase center of the 60S ribosome subunit [7,8]. The C-terminal domain (C-domain) binds to eRF3 [9][10][11][12], and this interaction increases the efficiency of translation termination [13,14].…”

Structure and dynamics in solution of the stop codon decoding N‐terminal domain of the human polypeptide chain release factor eRF1

“…40 The restraint violations were monitored after each cycle of refinement by the NMRest program. 24 2631 NOEderived distance restraints, 316 dihedral angles, and 69 RDCs were used in the calculation of the final ensemble (see Supporting Information Table S1). …”

“…Previously we determined the structure of the middle (M) and C-terminal domains of human eRF1 in solution. 24,25 Clear distinctions between the protein conformations in solution and in crystals were found for both the M-and the C-domains, especially for their functionally important regions. Some chemical shift assignments for a limited set of the signals (Ca, Cb, 1 HN, and 15 N) of the N-domain of human eRF1 have been reported 26 but the structure of the N-domain in solution was not determined.…”