Molecular mechanisms of substrate selection and protein folding on the ribosome
by Joerg Mittelstaet
Date of Examination:2012-06-19
Date of issue:2013-06-18
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Holger Stark
Referee:Prof. Dr. Ralf Ficner
Persistent Address:
http://dx.doi.org/10.53846/goediss-3890
Files in this item
Name:Joerg_Mittelstaet_PhD_ thesis.pdf
Size:2.73Mb
Format:PDF
The following license files are associated with this item:
Abstract
English
Aminoacyl-tRNA (aa-tRNA) is delivered to the ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Cognate codon-anticodon interaction induces rapid GTP hydrolysis and allows aa-tRNA to accommodate on the ribosome and take part in peptide bond formation. To investigate how conformational changes in the aa-tRNA contribute to the induced-fit mechanism of aa-tRNA selection, we monitored the timing and extent of transient tRNA distortions upon reading cognate or near-cognate codons. Despite rather different GTP hydrolysis rates, the rearrangements are similar on cognate and near-cognate codons, suggesting that the tRNA distortion alone does not provide a specific switch for the preferential activation of GTP hydrolysis in EF-Tu on the cognate codon. While selection of canonical cognate aa-tRNAs on the ribosome is rapid and efficient, recruitment of tRNAs carrying non-canonical amino acids can be disfavored at several stages. Binding of εNH2-Bodipy576/589-Lys-tRNALys to EF-Tu is strongly affected by the presence of the modified amino acid, but its incorporation on the ribosome occurs with speed and efficiency comparable to that of canonical lysine. The modified aa-tRNA is not significantly delayed at the accommodation gate and peptide bond formation is not rate-limiting for the incorporation, suggesting that ternary complex formation is the major bottleneck for the incorporation of non-canonical amino acids into proteins. Non-uniform translation elongation kinetics can affect many co-translational processes including protein folding. By pre-steady-state kinetic analysis of translation elongation on natural mRNAs, we find that translation is transiently paused at distinct sites, many of which (albeit not all) seem to be associated with rare codon clusters. By analysis of formation and decay kinetics of nascent peptides we find that chain elongation proceeds in a stochastic rather than sequential manner, which might provide a source of gene-intrinsic noise during protein expression. Co-translational protein folding is a general mechanism to prevent misfolding and aggregation of newly synthesized proteins. Here the co-translational folding of the N-terminal domain of protein PrmC was analyzed in real time by monitoring FRET between two fluorescently labeled amino acids incorporated at distinct positions of the polypeptide. The results indicate that folding of the N-terminal domain is an early event in the co-translational protein folding pathway, which is independent of the folding of the C-terminal domain.
Keywords: gene expression; protein biosynthesis; protein folding; ribosome; RNA