Interaction of eukaryotic initiation factors eIF1A and eIF4G with the ribosome and assembly of ribosomal initiation complexes on Picornavirus IRESs.

Abstract

Ribosomes serve as the platform for decoding the genetic information carried in mRNA and translating it into polypeptides; they are a major target for natural and synthetic antibiotics. Although fundamental to gene expression, many aspects of translation in eukaryotes are still not well understood, particularly translation initiation. The goal of my thesis was to yield insights of the interplay of mRNA, tRNA, cellular factors with the ribosome that are critical for the initiation of polypeptide synthesis. Specifically, my work focused on the interactions of translation initiation factors eIF1A and eIF4G with the 40S ribosomal subunit and the interactions of picornavirus RNAs with cellular factors that help them to bypass the regulation of canonical translation initiation. Initiation is the key rate-limiting stage in the eukaryotic translation process and more than 12 eukaryotic initiation factors (eIFs) are involved in this step. Most eukaryotic cellular mRNAs utilize the cap-dependent initiation pathway, in which the ribosomal preinitiation complex first binds to the ‘cap’ structure of the mRNA and then scans to select the AUG start codon to initiate the translation process. eIF4G and eIF1A are two factors that are required for steps in the canonical translation initiation process and they have distinct functions: eIF4G promotes attachment of mRNA to the ribosome and facilitates scanning by ribosomal complexes on the mRNA. eIF1A is involved in tRNA recruitment, scanning, start codon selection and subunit joining. Instead of eIF4F binding to the 5’-end of capped mRNA, some viral RNAs contain an internal ribosomal entry site (IRES) in their 5’UTR; this structure allows the viral RNA to recruit the 40S ribosomal subunit to an internal site on the mRNA independently of the cap and of the 5’-end of the mRNA. Four types of IRESs that have been identified in viral mRNAs can bypass one or all steps of the cap-dependent initiation pathway. Type 1 IRESs are found in picornaviruses such as poliovirus; type 2 IRESs are located in the 5’ UTRs of picornaviruses of the Cardiovirus and Aphthovirus genera, such as Encephalomyocarditis virus (EMCV) and Foot-and-mouth disease virus (FMDV). These two IRESs bind directly to eIF4G and eIF4A subunits of eIF4F, don’t need eIF4E, and can bypass the scanning step in initiation. Although they do share other steps with and use many of the same canonical factors as the cap-dependent initiation pathway, these IRESs also need IRES trans-acting factors (ITAFs) to fulfill their function. Different IRESs in the same family have different requirements for the ITAFs. The reason for this, and the mechanism by which ITAFs promote IRES function have both been obscure. To answer these questions, the specific aims of my thesis are: (1) To locate the position of eIF4G on the 40S ribosomal subunit; (2) To characterize the mechanism of action of eIF1A and to determine its location on the 40S ribosomal subunit; (3) To characterize aspects of the mechanism of translation initiation on Type 1 and Type 2 picornavirus IRESs. By using the technique of directed hydroxyl radical cleavage, we determined the positions of eIF4G/eIF4A on the ribosome, near the exit (E) site of the mRNA-binding channel, suggesting that these subunits of eIF4F are situated at the trailing edge of the ribosome and that eIF4A may unwind secondary structure in the cap-proximal region of the 5’UTR to promote “landing” on them by ribosomal preinitiation complexes. We positioned all domains of eIF1A on the 40S subunit and correlated these positions with the functions of eIF1A in promoting recruitment of initiator tRNA to the ribosomal peptidyl (P) site, preventing premature binding of elongator tRNA to the aminoacyl (A) site, coordinating sequential adoption by the 40S subunit of the ‘open’ conformation (to permit binding of mRNA) and the ‘closed’ conformation (on initiation codon recognition), and in contributing to the stability of ribosomal complexes during scanning. We determined that Type 2 IRESs bound to eIF4G adopt a compact side-by-side orientation, and that ITAFs promote this orientation. We also observed that eIF4G binds with similar orientations to domain V of Type 1 IRESs and to the J-K domain of Type 2 IRESs, suggesting that this factor plays a similar mechanistic role in initiation on Type 1 and Type 2 IRESs. Together, these results have yielded insights into how canonical initiation factors interact with the ribosome to promote the recruitment of mRNA, how they promote ribosomal scanning and how IRESs interact with ITAFs, a subset of initiation factors and ribosomal complexes to bypass early steps in the canonical eukaryotic initiation pathway.