A CRYO-EM Structure of a Minimal Translation Initiation System: Deletion in Self-Sufficient IRES Prevents Translocation

Abstract

Translation occurs on the ribosome, a ribonucleoprotein machine that exists in many copies per cell and is conserved among all forms of life. In canonical eukaryotic translation initiation, a suite of protein factors is required for many essential processes: for messenger RNA (mRNA) to bind the ribosome and to align itself with the translation initiation site, for the first aminoacylated tRNA to bind the start codon, for the ribosome to shift the mRNA downstream by one codon, and more. These processes occur on and near the mRNA's start codon immediately downstream of a 5' modified-nucleotide cap.In a departure from canonical catalytic and location requirements, some eukaryotes and many viruses encode regions of highly structured RNA that can initiate translation without a 5' cap and at least some initiation factors. These RNAs are called internal ribosomal entry sites, or IRESes. We study a type of IRES found in the Dicistroviridae virus class. This type of IRES initiates translation of the second of two gene clusters in the continuous viral RNA genome, and, uniquely, it requires no initiation factors to hijack eukaryotic host ribosomes.Here, we present a three-dimensional reconstruction of one such IRES--the Cricket Paralysis Virus intergenic IRES--bound to the eukaryotic yeast ribosome. The structure was obtained by cryo-electron microscopy and single-particle reconstruction techniques. This particular IRES sample is a mutant lacking three consecutive nucleotides in a loop region; it cannot undergo the initial translocation event, when mRNA shifts to allow a new aminoacylated tRNA into the A-site. We examine how the deletion prevents translocation, and thus how the intact IRES, a unique minimal translation system, might enable translocation, an essential part of initiating the universal translation process that is not wholly understood.