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      •   BUIR Home
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      • Bilkent Theses
      • Theses - Department of Molecular Biology and Genetics
      • Dept. of Molecular Biology and Genetics - Master's degree
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      POLR2A/RPB1 subunit of RNA polymerase II interacts with NTDMED14 containing core mediator complex to facilitate basal and activator driven transcription

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      Embargo Lift Date: 2021-01-17
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      Author(s)
      Jabbar, Javaid
      Advisor
      Cevher, Murat Alper
      Date
      2020-06
      Publisher
      Bilkent University
      Language
      English
      Type
      Thesis
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      Abstract
      The Metazoan Mediator is a 2-MDa protein complex that consist of 30 subunits, most of which are evolutionarily conserved from yeast to humans8. The maintenance and regulation of the cell is dependent on spatiotemporal control of RNA polymerase II (Pol II) mediated transcription as a result of intrinsic and extrinsic signals. Perturbations caused by the environment and genetics can alter the fate of the cells and can lead to many diseases such as cancer. The role of Mediator is critical in maintaining the cellular environment as it relays signal to RNA polymerase II to regulate homeostasis, cell growth, cell differentiation and development. Thus, it is essential to understand the mechanism by which Mediator regulates the expression of Pol II genes. We have utilized Multibac expression system to synthesize recombinant protein subcomplexes of Mediator and Pol II subunits to elucidate the interaction surface between core Mediator complex and RNA Polymerase II. Our data indicates that POLR2A (RPB1) subunit of Pol II interacts with ~84 kDa N terminal region of Med14 (NTD-Med14) containing core Mediator complex. Furthermore, we also show that other subunits of Pol II including POLR2C (RPB3), POLR2D (RPB4), POLR2E (RPB5), POLR2F (RPB6), POLR2G (RPB7), POLR2H (RPB8), POLR2I (RPB9) POLR2L (RPB10), POLR2J (RPB11) and POLR2K (RPB12) does not interact with core Mediator complex. The binding assay also demonstrates that the recombinant RPB1 subunit competes with endogenous Pol II for the interaction with core Mediator, forming a stable RPB1-core Mediator protein complex. The interaction between RPB1 subunit and NTD-Med14 containing core Mediator complex is independent of Med26. We propose a model for Pol II recruitment to the promoter by core Mediator complex which demonstrates that NTD-Med14 of Core Mediator complex interacts with RPB1 subunit of RNA polymerase II and recruits it to the promoter to facilitate basal and activated transcription.
      Keywords
      Mediator complex
      Multibac expression system
      RNA polymerase II
      RPB1
      NTD-Med14
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      http://hdl.handle.net/11693/53973
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