Project Details
Projekt
Probing the mechanisms of unnatural protein translation with an engineered genetic code and orthogonal translational pairs
Applicants
Professor Dr. Nediljko Budisa; Dr. Andrea C. Vaiana
Subject Area
Biochemistry
Term
from 2012 to 2018
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 207100805
In our previous work we employed chemically distinct proline analogs to study translation of single prolines and oligo-proline stretches in target sequences. A key result of this work was the discovery (under in vivo expression conditions) that oligo-proline stretches are differently translated with different analogs. In collaboration with groups of Rodnina and Wilson we used a series of proline analogs in order to understand the mechanism of elongation factor P (EF-P) in translation of oligo-proline stretches. The use of a set of descriptors (e.g. side chain sterics, inductive effects, pKa), enabled us to identify proline puckering as a dominant factor affecting relative translational rates. We further made progress in characterizing translation with orthogonal pairs and found that the commonly observed inefficiency of orthogonal translation is not exclusively due to the competition between the orthogonal tRNA and release factors (RF) but rather due to ribosomal stalling.Based on these encouraging results, we set out 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. This will be carried out in the context of the already established cooperation with P4 and P6 groups. 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 will continue our studies aiming to understand the role of stalling in translation with orthogonal pairs in collaboration with P6 (parameterization) and P7 (profiling). 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.
DFG Programme
Research Units
