| dc.contributor.advisor | Cramer, Patrick Prof. Dr. | |
| dc.contributor.author | Fianu, Isaac | |
| dc.date.accessioned | 2021-10-19T12:10:47Z | |
| dc.date.available | 2021-10-25T00:50:08Z | |
| dc.date.issued | 2021-10-19 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0008-5949-3 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-8866 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | Reconstitution of the Integrator complex and its interaction with paused transcription elongation complex of Pol II-DSIF-NELF | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Cramer, Patrick Prof. Dr. | |
| dc.date.examination | 2020-06-09 | |
| dc.description.abstracteng | The Integrator complex (INT) is the latest and largest multi-subunit protein complex to be added to the list of factors involved in RNA Polymerase II (Pol II) transcription. INT consist of 15 subunits with an estimated molecular weight of 1.5 MDa. It is recruited to Pol II during initiation or early elongation of transcription. It plays important roles in transcription regulation during early elongation including premature termination of some messenger RNAs (mRNAs). It has also been shown to terminate small nuclear RNAs (snRNAs), enhancer RNAs and some viral RNAs. Despite the important roles of INT in transcription, there is currently no protocol to reconstitute INT for in vitro biochemical and structural studies. Here, I used the baculovirus and insect cell recombinant protein expression system to reconstitute INT. Most subunits of INT express poorly and form oligomers when purified independently and it was not possible to co-express all 15 subunits. I therefore divided INT into 4 subcomplexes based on identified inter-subunit interactions. Namely, a 7-subunits core-INT, a 3-subunit cleavage module (CM), a 3-subunit cleavage module interacting module (CMIM) and INTS1/12 heterodimer. Subsequently I established purification protocols for all these subcomplexes or their stable interacting domains as in the case of INTS1/12 heterodimer. Negative stain EM reveals that the core-INT (INTS2/3/5/6/7/8-DDX26B) is monomeric showing that the oligomerization of the constituent subunits is circumvented in the core-INT. Further cryo-EM reconstruction of core-INT reveals a low-resolution doughnut shape for this subcomplex. The CM (INTS4/9/11) has a trilobal shape as revealed by a cryo-EM reconstruction at ~20 Å.
For the first time, I reconstituted the full INT in vitro from the purified subcomplexes by amylose affinity pulldown. The interactions between the subcomplexes were assessed by crosslinking mass spectrometry (XL-MS). XL-MS reveals many crosslinks between core-INT, CMIM, and INTS1/12 heterodimer while the CM has very few crosslinks to the other subcomplexes. This result suggests that the CM may be a loosely associated module of INT.
Furthermore, I showed for the first time the in vitro binding of INT to the paused elongation complex (PEC) of Pol II, DSIF (DRB sensitivity-inducing factor) and NELF (negative elongation factor). I characterized inter complex protein-protein interactions between INT and PEC by XL-MS. It emerges that INT interacts mostly with NELF and Pol II but not DSIF according to the crosslinks observed. The INTS1, INTS6, and INTS12 subunits of INT appear to be involved in most of these interactions with PEC. This work has created the foundation for biochemical and structural characterization of INT as a complex and INT in the context of Pol II transcription regulation. | de |
| dc.contributor.coReferee | Tittmann, Kai Prof. Dr. | |
| dc.subject.eng | Integrator complex | de |
| dc.subject.eng | Transcription | de |
| dc.subject.eng | Protein purification | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0008-5949-3-9 | |
| dc.affiliation.institute | Biologische Fakultät für Biologie und Psychologie | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2021-10-25 | |
| dc.identifier.ppn | 1774528959 | |