| dc.contributor.advisor | Rodnina, Marina Prof. Dr. | |
| dc.contributor.author | Peng, Bee-Zen | |
| dc.date.accessioned | 2019-09-10T10:30:59Z | |
| dc.date.available | 2019-09-10T10:30:59Z | |
| dc.date.issued | 2019-09-10 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0003-C1AF-E | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7634 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | The Role of EF-G in Translational Reading Frame Maintenance on the Ribosome | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Stark, Holger Prof. Dr. | |
| dc.date.examination | 2018-09-14 | |
| dc.description.abstracteng | Translation of an mRNA by the ribosome is the final step of gene expression. During translation initiation, the ribosome establishes the mRNA reading frame with the help of initiator tRNA binding to the start codon. This reading frame is maintained during the entire process of translation. The interactions between the codon-anticodon duplex and elements of the ribosome decoding site ensure tight binding of tRNAs to their respective codons and are essential for fast and correct decoding. However, during the tRNA–mRNA translocation step, the interactions between the mRNA-tRNA complex and the ribosome have to be disrupted to allow the movement of the ribosome along the mRNA. This is when reading frame maintenance faces the greatest challenge during the elongation. Ribosome slippage into an alternative reading frame usually leads to the synthesis of inactive, misfolded or even toxic proteins that increase not only the energetic cost of translation but also compromise the cellular fitness. Maintaining the translational reading frame is one of the most important task for the ribosome in the translation, but the mechanisms are poorly understood.
Here we examine the mechanism of reading frame maintenance using a fully-reconstituted translation system from Escherichia coli. We have selected an mRNA sequence that allows significant frameshifting and analyzed the roles of the ribosome and elongation factor G (EF-G) in this process. Based on crystal and cryo-EM structures of the ribosome–EF-G complexes, residues at the tip loops of domain IV of EF-G were replaced to examine the role of EF-G on reading frame maintenance. We show that the ribosome is highly prone for spontaneous frameshifting on a slippery sequence, whereas EF-G suppresses frameshifting. Single amino acid exchanges in key positions of domain IV of EF-G greatly increase frameshifting. Kinetic experiments indicate that the ability of EF-G to suppress spontaneous frameshifting correlates with the speed of translocation. Using the toolbox of fluorescence reporters, we identify how the trajectories of translocation and motions of the ribosome alter with the EF-G mutants. Our results suggest that the potential interactions between the residues at the tip of domain IV of EF-G and the mRNA-tRNA complex are essential during translation. Disruption of these interactions interferes with the dynamics of the SSU head and body domains movements, slow down the late translocation events, and open the kinetic window that allows the ribosome to shift into an alternative reading frame. Our work demonstrates the contribution of EF-G on reading frame maintenance during translocation. | de |
| dc.contributor.coReferee | Ficner, Ralf Prof. Dr. | |
| dc.subject.eng | ribosome; translocation; EF-G; reading frame maintenance | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0003-C1AF-E-9 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 1676455914 | |