| dc.description.abstract | Plants are constantly challenged with a variety of biotic and abiotic stress conditions. Being sessile, plants cannot evade these conditions. Most of these sudden changes in environmental conditions require a quick change in the expression of proteins involved in stress response. Translation is a critical process for rapid gene expression in the cell. Therefore, plants have evolved unique features in the components of protein synthesis machinery to facilitate a multifaceted response to the changing environmental conditions. Ribosomes, the core component of the translational machinery, have evolved with plant-specific features distinct from the fungal and metazoan ribosomes. A high-resolution structure of plant ribosomes would allow the visualization of these plant-specific features and their impact on the structure and function of the plant ribosome. Therefore, to gain structural insight into the unique features of plant ribosomes, we determined the structure of wheat ribosomes at high resolution.
We observed a high number of chemical modifications on rRNA in wheat ribosomes with plant-specific chemical modifications in the ribosome's active regions, such as PTC (Peptidyl Transferase Centre) and PET (Peptide Exit Tunnel). Additionally, we observed other plant-specific features, like the absence of zinc-finger motif in eL34 and extended uL4, making an exclusive interaction network. We note differences in the eL15-Helix 11 (25S) interaction, eL6-ES7 assembly and certain rRNA chemical modifications between monocot and dicot ribosomes. We observe highly conserved rRNA modification (Gm75) in eukaryotes in 5.8S rRNA and a flipped base (G1506) in PET, which is likely involved in sensing the nascent chain. Finally, we propose the importance of the universal conservation of three consecutive rRNA modifications in all ribosomes for their interaction with the A-site aminoacyl-tRNA.
Besides ribosomes, plants show distinct features in eukaryotic initiation factors (eIFs), especially the mRNA-recruiting eIF4 complex. The eIF4 group of factors include eIF4G (Scaffold Protein), eIF4E (mRNA cap-binding protein), eIF4A (helicase protein), and eIF4B (stimulation of mRNA helicase activity). Besides canonical eIF4G and eIF4E, plants possess unique isoforms, i.e., eIFiso4G and eIFiso4E. While eIF4F (eIF4G and eIF4E) binds structured mRNA, eIFiso4F (eIFiso4G and eIFiso4E) preferably binds to unstructured mRNA with monomethylated or hypermethylated cap region. The structural basis of this specificity is yet to be understood. However, attempts to study the structure of eIF4 proteins and complexes from other species like fungi and mammals have been challenging due to the large size and extensive intrinsic disorder in eIF4G. eIFiso4G is smaller with minimal intrinsic disorder and forms a high-affinity complex with eIFiso4E, making eIFiso4F more amenable for structural studies. Therefore, to understand the basis of mRNA specificity by eIFiso4F and the mechanism of mRNA activation by eIFiso4 complex (eIFiso4G, eIFiso4E, eIF4A and eIF4B), we aimed to purify and reconstitute eIFiso4F-mRNA complex for determining its structure by Cryo-EM. We purified and reconstituted a minimal eIFiso4F-eIF4A complex and performed cryo-EM, which resulted in a low-resolution map of the eIF4G-eIF4A complex. Further experiments are needed to improve the resolution and to obtain the density for eIF4E that would allow us to understand the mechanism of mRNA activation by eIF4.
Many plant viruses interact with and utilize eIF4 factors to translate viral mRNA. One such virus is BYDV (Barley Yellow Dwarf Virus), which infects crops like wheat, rice, barley maize, etc. It has structured 5'UTR and 3'UTR in the mRNA. The structured 3'UTR binds eIF4G and enhances the translation of viral mRNA by recruiting 40S in the presence of other helicase factors, eIF4A, eIF4B and ATP. Therefore, 3'UTR is also known as 3'BTE (BYDV Translation Enhancer Element). Biochemical studies suggest a large-scale conformational change to be crucial for recruiting eIF4 factors from 3'BTE to 5'UTR on the ribosome by long-range interaction between the two UTRs. Further, an extensive reorientation of eIF3 has also been proposed to be involved in translating BYDV mRNA. Hence, the translation initiation complex of BYDV mRNA undergoes a dramatic conformational change during protein synthesis. Understanding the structural basis of the translation initiation mediated by 3'BTE is essential to provide the mechanical details of the BYDV mRNA translation and for engineering BYDV-resistant varieties of crop plants. This prompted us to purify and perform cryo-EM studies on ribosomal complexes of plants with 3'BTE RNA. We obtained a cryo-EM map of an 80S ribosomal complex with P-site tRNA and mRNA in the decoding centre. However, the density for the structured 3’BTE is not visible, suggesting it is flexible. Also, the density for elongation factors (eEFs) was not seen. In another map, we observe eEF2 and eIF5A in 80S but no density for tRNA and mRNA. Further experiments with alternate strategies may be needed to obtain the density for the 3’BTE structural element on the 80S ribosomal complex.
Overall, this study provides structural insights into plant translation, focusing on the distinct features of plant ribosomes and the mRNA-recruiting eIF4 complex. | en_US |
