Abstract
Eukarya translation termination requires the stop codon recognizing protein eRF1. In contrast to the multiple proteins required for translation termination in Bacteria, eRF1 retains the ability to recognize all three of the stop codons. The details of the mechanism that eRF1 uses to recognize stop codons has remained elusive. This study describes the structural effects of mutations in the eRF1 N-domain that have previously been shown to alter stop codon recognition specificity. Here, we propose a model of eRF1 binding to the pre-translation termination ribosomal complex that is based in part on our solution NMR structures of the wild-type and mutant eRF1 N-domains. Since structural perturbations induced by these mutations were spread throughout the protein structure, residual dipolar coupling (RDC) data were recorded to establish the long-range effects of the specific mutations, E55Q, Y125F, Q122FM(Y)F126. RDCs were recorded on 15N-labeled eRF1 N-domain weakly aligned in either 5% w/v n-octyl-penta (ethylene glycol)/octanol (C8E5) or the filamentous phage Pf1. These data indicate that the mutations alter the conformation and dynamics of the GTS loop that is distant from the mutation sites. We propose that the GTS loop forms a switch that is key for the multiple codon recognition capability of eRF1.
Highlights
Eukaryotic protein synthesis is comprised of three stages namely, initiation, elongation and termination which results in the release of the nascent peptide
In an earlier report we demonstrated that the selectivity of stop codon recognition might be governed by the multiple conformations adapted by the strictly conserved GTS loop[23]
From cross-linking studies[26], the NIKS loop is assumed to interact with the first U of the stop codon via the anti-codon mimicry model proposed by Bertram et al.[10,33]
Summary
Eukaryotic protein synthesis is comprised of three stages namely, initiation, elongation and termination which results in the release of the nascent peptide. Based upon mutagenesis and sequence analysis, the N-domain is believed to be the site of codon decoding[7,8,18] This domain contains the highly conserved NIKS motif (residues 61–64), YxCxxxF motif (residues 125–131)[19] and the GTS loop (residues 31–33), which have all been implicated in stop codon recognition[9,15,20,21]. These elements are widely distributed throughout the protein structure confounding the identification of their precise roles in the stop codon recognition Conserved residues such as E55 and Y125 (Fig. 1), which are conserved in both eukaryotic and archeal release factors, have been implicated in models for eRF1 decoding site recognition. These observations further indicate that switching between omnipotency and unipotency in eRF1 may be modulated by distinct conformations of the GTS loop, which in turn are determined by the global structure of the N-domain and perhaps might be altered by the interactions with other components of the translation termination machinery
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