Dynamics of Stop Codon Discrimination by Release Factor 1

Abstract

During the elongation stage of translation, class I release factors (RFs) compete with aminoacyl-tRNAs to interrogate the mRNA triplet-nucleotide codon that is located in the ribosomal aminoacyl-tRNA binding (A) site. To avoid deleterious effects, class I RFs must be able to accurately discriminate stop codons from sense codons, only triggering the termination stage of translation and catalyzing the release of the nascent polypeptide chain from the peptidyl-tRNA located in the ribosomal peptidyl-tRNA binding (P) site upon recognition of a stop codon. Despite its importance for ensuring the accuracy of gene expression, the high fidelity mechanism through which class I RFs discriminate sense codons remains elusive. Using single-molecule fluorescence resonance energy transfer (smFRET), we have investigated the kinetics with which a fluorophore-labeled, bacterial class I RF (RF1) binds to the A site of bacterial ribosomal complexes carrying a fluorophore-labeled peptidyl-tRNA in the P site and either a stop codon, a sense codon that differs from a stop codon by a single nucleotide (i.e., a near-stop codon), or a sense codon that differs from a stop codon by two or more nucleotides at the A site. The results of these experiments, as well as analogous experiments performed using RF1 mutants or antibiotic inhibitors of RF1 function, reveal that RF1 discriminates sense codons from stop codons via a multi-step process that we are currently characterizing. Taken together with complementary molecular dynamics simulations of wildtype RF1 and RF1 mutants, our smFRET data are enabling us to elucidate the molecular details through which class I RFs discriminate the identity of the codon in the A site in order to ensure the integrity of translation elongation and the fidelity of translation termination.