| dc.contributor.advisor | Rodnina, Marina Prof. Dr. | |
| dc.contributor.author | Frister, Jan Ole | |
| dc.date.accessioned | 2019-01-25T11:17:57Z | |
| dc.date.available | 2019-11-27T23:50:02Z | |
| dc.date.issued | 2019-01-25 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-002E-E572-A | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7256 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 572 | de |
| dc.title | Recognition Elements for Elongation Factor P on the Ribosome | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Tittmann, Kai Prof. Dr. | |
| dc.date.examination | 2018-11-29 | |
| dc.description.abstracteng | Translation elongation is a multi-step process orchestrated by elongation factors. Elongation factors G and Tu are required for each round of translation elongation, whereas elongation factor P is only required to assist the translation of poly(Pro) sequences. Upon incorporation of consecutive proline residues the ribosome is stalled; this stalling is alleviated by EF P. While the catalytic mechanism of EF-P is well described, the determinants of EF-P binding remain unknown. Structural data and biochemical studies suggest the E-site codon, the peptidyl tRNA, the ribosomal protein L1 and the post-translational modification of EF-P as key interaction partners during binding and EF P assisted catalysis.
In this thesis we developed a FRET based EF-P binding assay using fluorescence-labeled ribosome complexes and a quencher-labeled EF-P. We combined the binding assay with different EF-P activity assays to determine the contribution of each of the proposed interactions to the binding and the catalytic activity of EF-P. We found that EF-P binds to different ribosome complexes with similar rates. EF-P has a short residence time on complexes without poly(Pro) stalling sequences, which is significantly increased on poly(Pro) stalled complexes. This high affinity state depends on the presence of several recognition elements in poly(Pro)-stalled complexes, in particular tRNAPro in the P site and the polypeptide chain containing several sequential proline residues. The context-independent association rates and the determined cellular concentration of EF-P suggest that the sampling of ribosome complexes by EF-P is kinetically controlled by the availability of a vacant E site. However, only poly(Pro)-stalled ribosome complexes provide the interactions required for the high-affinity binding of EF-P. The dissociation rates from Pro-stalled and non-stalled complexes match the reported rates of EF P assisted peptide bond formation. This suggests a mechanism in which the prolonged residence time for stalled complexes allows EF-P to position the peptidyl-tRNA in a catalytically active conformation and thereby to alleviate the stalling. After peptide bond formation the complex returns to the low affinity state, inducing dissociation of EF-P. The proposed kinetic regime allows EF-P to efficiently sample ribosomes with empty E sites, to recognize Pro-stalled complexes with high turnover rates and to alleviate stalling in a single functional cycle. Thus, our work demonstrates that the recruitment of EF-P is kinetically controlled contributing to a harmonized rate of translation. | de |
| dc.contributor.coReferee | Stark, Holger Prof. Dr. | |
| dc.contributor.thirdReferee | Ficner, Ralf Prof. Dr. | |
| dc.contributor.thirdReferee | Urlaub, Henning Prof. Dr. | |
| dc.subject.eng | Ribosome | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-002E-E572-A-5 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2019-11-27 | |
| dc.identifier.ppn | 1047415658 | |