Protocol for the in vitro reconstruction of site-specifically phosphorylated RNA Pol II to identify the recruitment of novel transcription regulators.
| Autor: | Hardtke HA; Department of Molecular Biosciences, University of Texas, Austin, TX 78712, USA. Electronic address: hhardtke@utexas.edu., Moreno RY; Department of Molecular Biosciences, University of Texas, Austin, TX 78712, USA., Zhang YJ; Department of Molecular Biosciences, University of Texas, Austin, TX 78712, USA. Electronic address: jzhang@cm.utexas.edu. |
|---|---|
| Jazyk: | angličtina |
| Zdroj: | STAR protocols [STAR Protoc] 2024 Sep 20; Vol. 5 (3), pp. 103277. Date of Electronic Publication: 2024 Aug 27. |
| DOI: | 10.1016/j.xpro.2024.103277 |
| Abstrakt: | The repetitive C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) becomes differentially phosphorylated throughout the transcription cycle. Here, we present a protocol to site-specifically phosphorylate the CTD of RNAPII by leveraging the specificity of well-characterized CTD kinases. We describe the steps for optimal phosphorylation of the CTD and the preparation of nuclear protein extract. This protocol can be used to identify the interactome of a phospho-CTD and has the potential to identify novel RNAPII-binding proteins. For complete details on the use and execution of this protocol, please refer to Moreno et al. 1 . Competing Interests: Declaration of interests The authors declare no competing interests. (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
| Externí odkaz: |
|