Abstract
Transcription in eukaryotes is carried out by RNA polymerases, (Pol) I, II and III. RNA polymerases are structurally and functionally well characterised, consisting of 12-17 subunits that assemble into a complex of over 500 kDa, but their biogenesis and nuclear import remain poorly understood. Iwr1, a protein identified to bind Pol II in yeast, was identified as a Pol II nuclear import factor and may also play a role in transcription. Iwr1 is functionally conserved across eukaryotes as knockdown of the Iwr1 homolog SLC7A6OS in zebrafish results in severe neurodevelopmental defects and in humans, splice variants of SLC7A6OS cause progressive myoclonus epilepsy.Early work determined the architecture of the Iwr1-Pol II by cryogenic-electron microscopy (cryo-EM) at 21 Å resolution prior to the ‘resolution revolution’. Cryo-EM densities attributed to Iwr1 were observed within the Pol II active site formed between its two largest subunits and the Pol II stalk. However, given the resolution obtained, these densities are insufficient for an atomic model, or may even be artefactual. So key questions remain regarding the biochemical basis of the Iwr1-Pol II interaction. Here, we aimed to solve the structure of Iwr1-Pol II to allow atomic modelling of their interactions and provide mechanistic insight into Pol II assembly and nuclear import. Several attempts at cryo-EM using BS3 cross-linked Iwr1-Pol II led us to a structure at 3.4 Å, however an Iwr1 density could not be attributed to the active site of Pol II. Further work may aim to use different, non- specific cross-linkers such as glutaraldehyde with gradient fixation to solve the Iwr1-Pol II structure.
| Date of Award | 3 Oct 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Alan C M Cheung (Supervisor) |