Free energy landscape of RNA binding dynamics in start codon recognition by eukaryotic ribosomal pre-initiation complex.

Takeru Kameda,Yuichi Togashi,Katsura Asano

doi:10.1371/journal.pcbi.1009068

Abstract

Specific interaction between the start codon, 5'-AUG-3', and the anticodon, 5'-CAU-3', ensures accurate initiation of translation. Recent studies show that several near-cognate start codons (e.g. GUG and CUG) can play a role in initiating translation in eukaryotes. However, the mechanism allowing initiation through mismatched base-pairs at the ribosomal decoding site is still unclear at an atomic level. In this work, we propose an extended simulation-based method to evaluate free energy profiles, through computing the distance between each base-pair of the triplet interactions involved in recognition of start codons in eukaryotic translation pre-initiation complex. Our method provides not only the free energy penalty for mismatched start codons relative to the AUG start codon, but also the preferred pathways of transitions between bound and unbound states, which has not been described by previous studies. To verify the method, the binding dynamics of cognate (AUG) and near-cognate start codons (CUG and GUG) were simulated. Evaluated free energy profiles agree with experimentally observed changes in initiation frequencies from respective codons. This work proposes for the first time how a G:U mismatch at the first position of codon (GUG)-anticodon base-pairs destabilizes the accommodation in the initiating eukaryotic ribosome and how initiation at a CUG codon is nearly as strong as, or sometimes stronger than, that at a GUG codon. Our method is expected to be applied to study the affinity changes for various mismatched base-pairs.

Highlights

The translation reaction, or mRNA-dependent protein synthesis, is catalyzed by the ribosome, the macromolecular ribonucleoprotein complex [1, 2]
The start codon base-pairing with the Met À tRNAMi et anticodon in the P-site allows the 40S subunit to stall at the start codon and join the 60S subunit after most bound eukaryotic initiation factors (eIFs) are released in conjunction with Pi release from eIF2 [7, 8]
Since we did not observe a strong disorder in CUG-anticodon structure (Fig 7), we propose that the near-cognate start codon usage characteristic of eukaryotic initiation is mostly explained by a strong perturbation on GUG accommodation in the P-site due to steric restriction imposed by eIF1 β-hairpin loop

Summary

Introduction

The translation reaction, or mRNA-dependent protein synthesis, is catalyzed by the ribosome, the macromolecular ribonucleoprotein complex [1, 2]. The ribosome dissociates into the large (60S) and small (40S) subunits, and the latter binds the methionyl initiator tRNA (Met À tRNAMi et) and mRNA with the help of eukaryotic initiation factors (eIFs) [3, 4]. Met À tRNAMi et is recruited by eIF2, a heterotrimeric factor that binds the tRNA in a manner dependent on GTP binding. The resulting ternary complex (TC) binds the 40S subunit in the context of multifactor complex (MFC) with eIFs 1, 3 and 5, forming the 43S preinitiation complex (PIC) [5]. The 48S pre-initiation complex (PIC) formed scans for the start codon in the process called scanning. The resulting 80S initiation complex accepts an amino-acyl tRNA in the Asite to begin the translation elongation cycle

Methods

Results

Discussion

Conclusion