dc.contributor.advisor | Rodnina, Marina Prof. Dr. | |
dc.contributor.author | Sharma, Heena | |
dc.date.accessioned | 2017-07-25T08:18:35Z | |
dc.date.available | 2017-07-25T08:18:35Z | |
dc.date.issued | 2017-07-25 | |
dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0023-3EB3-C | |
dc.identifier.uri | http://dx.doi.org/10.53846/goediss-6407 | |
dc.language.iso | eng | de |
dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
dc.subject.ddc | 572 | de |
dc.title | Kinetics of subunit rotation of the ribosome during tRNA-mRNA translocation | de |
dc.type | doctoralThesis | de |
dc.contributor.referee | Tittmann, Kai Prof. Dr. | |
dc.date.examination | 2016-11-07 | |
dc.description.abstracteng | 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. | de |
dc.contributor.coReferee | Stark, Holger Prof. Dr. | |
dc.subject.eng | Translation elongation | de |
dc.subject.eng | EF-G | de |
dc.subject.eng | Ribosome | de |
dc.subject.eng | Subunit rotation | de |
dc.subject.eng | Kinetics | de |
dc.subject.eng | FRET | de |
dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0023-3EB3-C-0 | |
dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
dc.subject.gokfull | Biologie (PPN619462639) | de |
dc.identifier.ppn | 894255762 | |