1. mRNA scanning by mammalian ribosomes
In eukaryotes protein synthesis starts with the recruitment of the small ribosomal subunit to the capped mRNA 5’ end. The small subunit then moves towards the mRNA coding region in search for the correct AUG start codon. This process is called mRNA scanning. Codon recognition triggers the binding of the large ribosomal subunit and with that the formation of a complete translation competent ribosome complex.
Scanning and start codon recognition are tightly regulated steps in gene expression, where the reading frame of each protein is defined and the translation frequency of each gene is fine tuned. However, the molecular details of these processes are largely unknown. We employ single molecule fluorescence spectroscopy methods to understand the principle mechanism of scanning, its relation to the structural dynamics of the ribosome and its regulation by translation factors.
2. RNA translocation
tRNAs are adaptor molecules that couple the correct amino acid to the respective codon encoded by the mRNA. During translation they have to pass the three tRNA binding sites on the ribosome (the acceptor, peptidyl and exit site). The process of tRNA movement through the ribosome is called translocation. In bacteria translocation is catalyzed by a ribosomal co-factor, the GTPase elongation factor G (EF-G).
Using single molecule FRET and TIRF microcopy we recorded tRNA trajectories through the ribosome during EF-G catalyzed translocation. We were able to show that tRNAs adopt intermediate states on the ribosome which we call chimeric states. We assigned these states to published structures of EF-G-ribosome-complexes and -supported by biochemical and kinetic experiments- placed them on the translocation reaction coordinate.
In a follow-up project we are now interested in translocation events leading to erroneous peptide sequences. Deviations from the correct sequence potentially lead to misfolded proteins. These tend to form aggregates which are toxic to cells and might cause diseases. Our work aims to generate a refined and comprehensive mechanistic model for canonical and non-canonical tRNA translocation events.
3. Rotation dynamics during translation termination
Efficient protein synthesis requires the exact coordination of intermolecular ribosome motions. Ribosome interaction with its ligands (translation factors or tRNAs) modifies the pattern of motion in order to optimize it for each particular task. The rotational motion of the small ribosomal subunit with respect to the large subunit is a key dynamic element that is important in all states of translation. We investigated the role of subunit rotation for the termination step.
Termination of protein synthesis starts when the ribosome encounters a stop codon signaling the end of protein synthesis. Then termination factors bind to the ribosome and release the nascent peptide. We generated FRET labels on various parts of termination complexes to define the role of ribosome dynamics for the termination process and to study the interplay of termination factors with the ribosome. We gained a multi-perspective view on the termination pathway that allowed us to generate a comprehensive mechanistic model.
In future projects we will focus on non-canonical termination events that occur on truncated mRNAs in a stop codon independent manner. Here, a special termination machinery is recruited that releases premature peptides and makes them available for degradation. We will use single molecule fluorescence techniques to solve the molecular details of the reaction scheme.