Interrogating the Mechanism of Eukaryotic Initiation Factor 3 (eIF3) through Molecular Dissection

Abstract

Eukaryotic translation initiation is the rate‐limiting and most regulated step of protein synthesis. It begins with the formation of a pre‐initiation complex (PIC) on the small (40S) ribosomal subunit. The PIC is then recruited to the 5′ end of an mRNA, from which it scans in the 5′ to 3′ direction to identify the start (AUG) codon. This convoluted pathway is mediated by multiple protein factors called eukaryotic initiation factors (eIFs).eIF3, the largest and the most complex of these protein factors, is comprised of five essential subunits (eIF3a/b/c/g/i). Previous genetic and biochemical studies have shown that eIF3 mediates PIC formation, is essential for mRNA recruitment, and participates in scanning for the start codon. eIF3 has also been shown to play a critical role in the stabilization of mRNA in the exit channel and contributes to the rate of recruitment at the entry channel. This latter role of eIF3 may involve its interaction with the 40S latch elements Rps2/3 and h16/h18. In fact, mutations of Rps3 exacerbate 40S dependence on eIF3 for stabilizing mRNA binding by the PIC. Using an in‐vitro reconstituted system, we are interrogating the interaction between eIF3 and the 40S latch by combining Rps2/3 mutations and mutations within elements of eIF3 thought to interact the latch.To further dissect the role of the eIF3 entry and exit‐channel arms, we are reconstituting yeast eIF3 by recombinant expression in bacteria. Previous work has relied on eIF3 purified from yeast cells. This new approach will alleviate the limitations previously imposed by the essential nature of the eIF3 subunits. Our initial work will examine the activity of reconstituted eIF3 in mRNA recruitment and PIC stabilization. Then, we will focus on a set of mutations targeted at interrogating the many functions of eIF3: truncation of a region of eIF3c thought to promote accurate start‐codon selection; isolation of the eIF3a/c heterodimer that stabilizes mRNA at the ribosomal exit channel; isolation of a eIF3b‐g‐i complex found near the mRNA entry channel; and truncation of an RNA binding domain of eIF3b. The effects of these mutations on component events during translation initiation will shine new light on the role of eIF3 and its subunits.