Ribosome Processivity and Co-translational Protein Folding
by Michael Sebastian Thommen
Date of Examination:2015-11-03
Date of issue:2017-04-21
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Heinz Neumann
Referee:Prof. Dr. Marina Bennati
Persistent Address:
http://dx.doi.org/10.53846/goediss-6242
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Abstract
English
Synthesis of polypeptide chains on the ribosome occurs at a non-uniform rate. mRNA sequences translated at a high rate are interspersed by segments that lead to translational pausing. The rate of translation elongation is rate-limiting for conformational sampling during co-translational folding of the nascent chain. The global and local rates of translation elongation are important modulators of the efficiency of co-translational folding. For the understanding of co-translational folding, a description of the dynamics of the nascent chain synthesis has to be established in the first place. To understand how tRNA pools affect local and global translation velocities, we have constructed a mathematical stochastic model based on the availability and competition of tRNA isoacceptors for the A site. We predicted the elemental rate constants for the decoding steps in vivo, which allowed us to calculate codon-specific translation elongation rates for any mRNA sequence. We then have analyzed the pausing during translation of natural mRNA in vitro. The accumulation of translation intermediates upon synthesis of the model protein HemK could not be explained by the propensity of mRNA to form local stable secondary structures or by the usage frequency of codons. We have established novel experimental approaches that might allow the identification of the site of translational pausing with single codon resolution. To investigate co-translational folding in real-time, we developed tools to incorporate FRET probes at internal positions of nascent chains mediated by tRNACys. We observed that peptide bond formation was limited by substrate positioning in the peptidyl-transferase center of aminoacyl-tRNA carrying larger fluorophores. Glycine residues upstream of the incorporation site tuned the incorporation of larger fluorescent probes by increasing the flexibility of the peptidyl-tRNA in the P site. The interplay between nascent chain elongation and co-translational folding was studied using the model protein γ-B-crystallin encoded by the original bovine sequence and a sequence harmonized for Escherichia coli codon usage. We observed differences in local and global translation elongation for both mRNA sequences. By applying a pulse proteolysis approach and the incorporation of FRET pairs in the nascent chain, we detected altered kinetics of co-translational folding for both domains of γ-B-crystallin depending on the mRNA sequence. In summary, we show how changes in local translation elongation can influence the acquisition of folded states in the nascent chain.
Keywords: ribosome