5. Nediljko Budisa/Andrea Vaiana - Probing the mechanisms of unnatural protein translation with an engineered genetic code and orthogonal translational pairs

We aim to fully elucidate the mechanism of EF-P in protein synthesis by using chemically distinct proline analogs in target sequences and molecular dynamics simulations. First, we will generate a chemical peptide model to measure the rate of amide bond formation with various analogues and we will compare these rates with ribosomal rates. Furthermore, we will perform extensive molecular dynamics simulations of the ribosome EF-P complex to further investigate the mechanism by which poly-proline stretches induce stalling in bacterial ribosomes and how this stalling is resolved by EF-P. We plan to pinpoint the location of stalled ribosomes on the cellular mRNA pool in presence or in absence of RF 1. Further characterization of orthogonal pair efficiency will be conducted via our reporter gene activity and quantification assays in the presence of a different number of in-frame amber stop codons. In addition, we will use pyrrolysine (Pyl)-based o-pairs (PylRS:tRNAPyl) combined with distinct modification analogs to elucidate the role of the ß lysyl-Lys34 modification in EF-P. Incorporation of crosslinking analogs and optimized orthogonal translation setups resulting from our work will provide new, efficient tools for future structural as well as co-translational folding studies.