Insights into mRNA recruitment by eukaryotic initiation factor 4 during translation in Saccharomyces cerevisiae
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Ribosomes are responsible for synthesizing proteins in the cells by using mRNA as the template. However, the ribosomes cannot perform this function without the help of other protein factors. Different factors are required at all the stages of translation – initiation factors during translation initiation, elongation factors during elongation, and termination factors are required for translation termination. Recycling factors then help in the dissociation of the ribosome into its large and small ribosomal subunits. Of all the stages, initiation is a highly evolved, regulated, and complex process in eukaryotes compared to their prokaryotic counterparts. During initiation, mRNA is recruited to the small ribosomal subunit and is followed by recognition of the start codon. The Association of the large ribosomal subunit marks the end of initiation and results in the formation of the elongation competent ribosome. In prokaryotes, the base pairing of the Shine-Dalgarno sequence in mRNA with the anti-Shine-Dalgarno sequence present in the 16S rRNA leads to mRNA recruitment and places the start codon at the P-site. However, in the case of eukaryotes, there is a specialized group of factors called eIF4 that help in mRNA recruitment to the 40S. Studying the function of these eIF4 proteins in mRNA recruitment is the focus of this thesis. eIF4B is one factor of the eIF4 group that is essential for the translation of mRNAs. Initial studies on this factor reported that it merely plays a stimulatory role during initiation, where it enhances the mRNA secondary structure unwinding activity of the RNA helicase factor eIF4A. However, there are also reports which show direct interaction of eIF4B with the 40S in yeast. To gain more insights into the roles played by eIF4B during initiation in yeast, we used cryo-EM to study the structure of the 40S-eIF4B complex. In the 4.8Å structure of 40S-eIF4B, we observed that eIF4B is located on the solvent-exposed side of the 40S and near the mRNA entry channel. Along with this, the mRNA channel latch is partially open when compared to the closed latch structure of the 40S. This suggests that eIF4B may be helping in mRNA recruitment by remodeling the 40S mRNA channel latch. When compared to the 43S PIC structure, we observe a steric clash of eIF4B with eIF3j and eIF3g-RRM. This clash may be responsible for the dissociation of eIF3j as well as the relocation of the eIF3b-g-i complex to the subunit interface of the 40S. With this study, we could decipher the location of eIF4B on the 40S, its interacting partners, and its contribution to the overall dynamics of translation initiation. eIF4G & eIF4E are other factors of the eIF4 group where eIF4E recognizes the 5ʹ cap of the mRNA and eIF4G is the scaffolding protein responsible for keeping the entire eIF4 complex together. eIF4G also interacts with factors of the 43S PIC, thus helping in mRNA recruitment. This complex is also tightly regulated by the eIF4E and eIF4G binding proteins, keeping a check on mRNA recruitment. This regulation forms the basis for embryonic development, differentiation, aging, and the response of cells to stress conditions. Even after being important, limited structural knowledge of the eIF4G-eIF4E complex exists. This is mainly due to the presence of intrinsically disordered regions in eIF4G. In this work, we overexpressed & purified the S. cerevisiae eIF4G-eIF4E from E. coli and used cryo-EM to generate a 3D map of the complex. The complex appears to be L-shaped, however, the resolution is low owing to the conformational heterogeneity of eIF4G. Covalent cross-linking was tried to stabilize the complex, however, it resulted in aggregation of the sample during grid preparation. Overall, this study sheds light on the structural envelope of the eIF4G-eIF4E complex and highlights the technical challenges of studying the structure of this complex.