Kinetics of subunit rotation of the ribosome during tRNA-mRNA translocation
von Heena Sharma
Datum der mündl. Prüfung:2016-11-07
Erschienen:2017-07-25
Betreuer:Prof. Dr. Marina Rodnina
Gutachter:Prof. Dr. Kai Tittmann
Gutachter:Prof. Dr. Holger Stark
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-6407
Dateien
Name:PhD Thesis.pdf
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Zusammenfassung
Englisch
Ribosome dynamics play an essential role in orchestrating all stages of protein synthesis. Recent biochemical, structural and computational studies have shown large-scale conformational changes of the ribosome, its tRNA substrates and translation factors during the elongation phase of protein synthesis. Dynamic movements of the ribosome not only govern the translation process but are also targeted by many antibiotics resulting in inhibition of protein synthesis. Therefore, complete understanding of conformational rearrangements in the ribosome will improve our knowledge about translation mechanism and its regulation which will also help to design novel antibiotics. One of the key dynamic processes important for ensuring forward movement of the tRNA-mRNA complex during translocation is the rotation of the small subunit (SSU) of the ribosome relative to the large subunit (LSU). Ribosomal subunits rotate spontaneously, i.e. in the absence of auxiliary translation factors, in the counterclockwise (CCW) direction upon the formation of deacylated tRNA in the P site of the ribosome as a result of peptide bond formation. Elongation factor G (EF-G) promotes the tRNA-mRNA translocation at the cost of GTP hydrolysis, which is accompanied by the clockwise (CW) rotation of the SSU. However, the exact role of subunit rotation in translocation is not properly understood. In this thesis, I present the real time kinetics of spontaneous subunit rotation and show how EF-G promotes and coordinates the rotation of the subunits with the movement of the tRNAmRNA complex along the ribosome. We used ribosomal subunits labeled with fluorescence reporters forming a FRET (Förster resonance energy transfer) pair and monitored the kinetics of subunit rotation relative to peptide bond formation and translocation using ensemble kinetics and single-molecule FRET (smFRET). We observed that spontaneous rotation of the SSU in CCW direction is rapid and reversible and is independent of the rate of preceding step of peptide bond formation. EF-G binding to the ribosome in the non-rotated state accelerates CCW rotation of the SSU by 5-fold. The transition back of the SSU body in clockwise (CW) direction to the non-rotated state starts early on the translocation pathway and precedes CW movement of the SSU head but overall coincides kinetically with the tRNA-mRNA translocation. The uncoupling of the movement of body and head of the SSU results in unlocking of the ribosome that allows translocation of the tRNA-mRNA complex. In addition, we show how the smooth synchronized motion of the SSU body and head can be perturbed by diverse antibiotics. Our work demonstrates how large-scale thermally driven movements of the ribosome are gated by its ligands such as EF-G, tRNAs and antibiotics.
Keywords: Translation elongation; EF-G; Ribosome; Subunit rotation; Kinetics; FRET
