Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons.

Kazuki Saito,Koichi Ito

doi:10.1093/nar/gkv376

Abstract

In eukaryotes, the tRNA-mimicking polypeptide-chain release factor, eRF1, decodes stop codons on the ribosome in a complex with eRF3; this complex exhibits striking structural similarity to the tRNA–eEF1A–GTP complex. Although amino acid residues or motifs of eRF1 that are critical for stop codon discrimination have been identified, the details of the molecular mechanisms involved in the function of the ribosomal decoding site remain obscure. Here, we report analyses of the position-123 amino acid of eRF1 (L123 in Saccharomyces cerevisiae eRF1), a residue that is phylogenetically conserved among species with canonical and variant genetic codes. In vivo readthrough efficiency analysis and genetic growth complementation analysis of the residue-123 systematic mutants suggested that this amino acid functions in stop codon discrimination in a manner coupled with eRF3 binding, and distinctive from previously reported adjacent residues. Furthermore, aminoglycoside antibiotic sensitivity analysis and ribosomal docking modeling of eRF1 in a quasi-A/T state suggested a functional interaction between the side chain of L123 and ribosomal residues critical for codon recognition in the decoding site, as a molecular explanation for coupling with eRF3. Our results provide insights into the molecular mechanisms underlying stop codon discrimination by a tRNA-mimicking protein on the ribosome.

Highlights

Stop codons are decoded by protein factors called class 1 and class 2 polypeptide-chain release factors (RFs) [1]
We found that a simple rule applied to sub-specific patterns of position123 residues as well as that there is a functional coupling of the position-123 residue of eRF1 with endogenous promoters of SUP35 (eRF3), which was not observed among neighboring residues, suggesting that the position-123 residue belongs to a novel class of functional residues in eRF1 involved in stop codon discrimination
A series of yeast expression plasmids encoding S. cerevisiae eRF1 (Sc-eRF1) mutants, containing single point mutations of all amino acid residues other than wild-type leucine at position-123, were constructed

Summary

Introduction

Stop codons are decoded by protein factors called class 1 and class 2 polypeptide-chain release factors (RFs) [1]. Class 1 RFs are proteins that functionally and structurally mimic tRNAs, while class 2 RFs are members of the translational GTPase family. Class 1 RFs enter the ribosomal A site to recognize stop codons and to stimulate hydrolysis of peptidyl-tRNA, thereby releasing the nascent peptide from the ribosome with the aid of the universally conserved Gly-Gly-Gln (GGQ) motif [1]. There are two dual-codonspecific class 1 RFs in eubacteria. After release of the nascent polypeptide, a class 2 RF, RF3, binds to the ribosome and indirectly facilitates the dissociation of RF1/2 from the ribosome in a GTPase-dependent manner [5]

Methods

Results

Conclusion