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Projekt
Stochastic modelling of protein synthesis by ribosomes
Antragsteller
Professor Dr. Reinhard Lipowsky
Fachliche Zuordnung
Biophysik
Förderung
Förderung von 2012 bis 2020
Projektkennung
Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 207100805
In the previous funding period, we developed a detailed theory for the kinetics of protein synthesis by ribosomes and introduced a new computational method to deduce the transition rates in vivo from their in-vitro values. In the next funding period, we intend to apply our theory to several systems as studied experimentally within the projects P6 (Rodnina) and P8 (Kramer and Bukau). Three specific goals will be addressed: mechanisms for translational pauses (with P6); peptide movement within the exit tunnel (with P6); and kinetics of co-translational protein assembly (with P8).The first goal is to further improve our analysis of the synchronized translation system studied in P6. Using this system, one can measure the time-dependent elongation of the peptide chains. The corresponding gels often exhibit several time-persistent bands. A preliminary analysis of these bands has shown that a certain fraction of the ribosomes stalls at certain codons or codon stretches and that these ribosomes resume their translation activity with a relatively low rate. Thus, when the ribosomes arrive at these codons, they can follow two different pathways, one of which involves a long-lived pausing state. In order to elucidate the nature of these latter states, we will systematically analyze the composition of the slow codon stretches and compare codon-specific and context-dependent mechanisms.The second specific goal is to determine the movement of the nascent peptide chain through the exit tunnel as measured in P6 by fluorescent labeling. By varying the length of the codon sequence, one can deduce codon-specific elongation rates and a possible contextdependence of these rates in an iterative manner. The dependence on codon context may arise from adjacent upstream codons for prolin, from 3-codon motifs that contain two adjacent prolin codons, and for longer motifs that interact with the peptide exit tunnel. One example for the latter motifs are peptide chains with positively charged residues. Because these fluorescence measurements in the exit tunnel are performed for the same buffer conditions as the synchronized translation system, the two experiments must be governed by the same transition rates which provides a nontrivial constraint for our theory.Our third specific goal is to address the kinetics of co-translational protein assembly as studied experimentally in P8. We will focus on luciferase, which is assembled from LuxA and LuxB subunits. The available data provide evidence that the assembly of these two subunits is facilitated by co-localized synthesis. Based on the experimental observations, we will extend our model to include the kinetics of co-translational folding, subunit diffusion in the cytosol, and interactions with the chaperone trigger factor. We will supplement these stochastic models by molecular dynamics simulations in order to elucidate the binding free energy of the two subunits
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