Comprehensive Understanding of Kozak Sequence Recognition on the Human Ribosome – Kozak

I have started the project by optimizing the production of endogenous complexes on different mRNAs in terms of reproducibility, amounts and quality of cryo-EM grids for structural studies. First high-resolution data was collected on the state-of-the-art G4 Titan Krios that was recently installed at the CBI/IGBMC
Further we have started to purify human initiator tRNA from HeLa cells and to analyze the purity as well as the chemical modifications of tRNAi by mass spectrometry. The tRNA will be used for structural studies of in vitro reconstituted complexes.
Finally we have started the development of the functional in vivo functional assays.

- We obtained first data for the perfect Kozak sequence In which we can observe cler signal for the mRNA as well as positioning of side chains of the initiation factors involved in Kozak sequence recognition. Structural interpretation of this data is ongoing.

- We have recently recruited a student to work on the in vitro reconstitution of translation initiation complexes. She was successful in obtaining human initiator tRNA and is working on upscaling the purification.

- We are in process to develop a pipeline to interpret the functional in vitro data.

- Structural studies will now be extended towards mRNA and eIF1A mutants as well as approaches to reconstitute the complex in vitro.
- Functional studies will be extended to analyse more globally the impact of mRNA and eIF1A mutants.

.

Eukaryotic protein synthesis depends on the correct AUG start codon recognition during translation initiation. This is particularly efficient for mRNAs that contain a strong Kozak sequence defined by specific nucleotides flanking the AUG start codon, but the underlying molecular mechanism, e.g. how eukaryotic initiation factors or ribosomal components recognize the Kozak sequence, is unknown. I have obtained cryo-EM structures of human translation initiation complexes with and without eukaryotic initiation factors at ~3Å resolution. These structures give insights into the molecular details by which the ribosome, eukaryotic initiation factors 1A and 2 are involved in Kozak sequence recognition and providing information on the molecular interactions of the Kozak sequence with the ribosome that can be used to design functional and structural analysis of Kozak mRNA complexes. In this project I want to further characterise the molecular mechanism of Kozak sequence recognition by extending the analysis towards (i) using and developing further the latest cryo-EM and image processing technologies to obtain a high-resolution structure of a key human 48S initiation to better resolve all possible factors involved in interactions with the mRNA, the 40S small ribosomal subunit and the initiator tRNA (ii) introducing point mutations into the mRNA Kozak sequence or the initiation factors involved in its recognition and analysing the effect of these mutations by structural and functional assays. This study will help to understand the role of eIFs in distinguishing between Kozak consensus and sequence variants and will reveal how start codons are selected during translation initiation in humans. For this I plan to produce a combination of structural and functional results consisting of high-resolution cryo-EM structures of initiation complexes with Kozak sequence mutants, and their functional contribution in in vitro and in vivo translation assays, polysome profiling and toe-printing. Studying the impact of the introduced mutations in the Kozak sequence and in the eukaryotic initiation factors will allow to make an accurate and global contribution analysis of the interactions that are necessary for Kozak sequence recognition during translation initiation. My results will be relevant for the general understanding of translation in humans, but also for cloning strategies, and may have applications in gene therapy and mRNA-based therapeutics.