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Freie Universität Berlin

1. Huang, Yong-Heng. Structure-based mechanisms of processive transcription anti-termination.

Degree: 2021, Freie Universität Berlin

Transcription is the initial step of gene expression in all domains of life. Antagonizing between termination and pre-mature transcription prevention (anti-termination) is a key aspect for gene expression regulation. λ phage protein N mediated anti-termination and ribosomal RNA anti-termination are two model processive anti-termination processes, both of which include NusA, NusB, NusE, NusG and additional factors. Although the topic has been investigated for more than 30 years, research have so far failed to uncover the structural basis for the complete transcription anti-termination complexes (TAC) and the detailed molecular mechanisms for antitermination. To address these aspects, here we report: (i) a cryo-electron microscopy (cryo-EM) structure of the entire λN-TAC, which contains a nucleic acid scaffold harboring nut RNA and an artificial transcription bubble, all Nus factors and λN; (ii) a crystal structure of the novel rrnTAC member SuhB complexed with the AR2 domain of NusA; and (iii) cryo-EM structures of rrnTACs composed of all Nus factors, SuhB with or without r-protein S4 on a rrnG leader region regulatory RNA (rrnGnut). Additionally, we applied biochemistry to clarify the basic mechanisms of the two anti-termination processes. The λN protein appears to have an “all-purpose” role in the λN-TAC. Firstly, it repositions NusA and remodels the β-subunit flap tip, by which the pause and termination hairpin formation and stabilization are disturbed. Secondly, by contacting upstream DNA in one side and holding it close to NusG, λN supports anti-backtracking. Third, λN helps in maintaining the elongation competent state of RNAP by invading and contacting along the hybrid cavity and RNA exit channel with its flexible C-terminus. Furthermore, ρ-dependent termination is counteracted due to the λN-induced repositioning of NusA and NusE, which sequesters the NusG C-terminal domain. The functional contributions of the novel rrnTAC member, SuhB, are currently unclear. With analytical size exclusion chromatography (analytical SEC), we show SuhB directly binds rrnGnut RNA at boxA-boxC spacer region and complexes with NusA stably. AR2 domain of NusA is identified to be the major binding platform for SuhB by further investigation. NusA and rrnGnut are required for SuhB to be successfully recruited to rrnTAC, while in turn SuhB is necessary for NusB/E integration. In vitro transcription assays revealed that SuhB is crucial for delaying or suppressing ρ-dependent termination as well as intrinsic termination. We also determined the crystal structure of SuhB-AR2 complex to elucidate the atomic basis. In vitro transcription using structure-guided mutations revealed the SuhB-AR2 interaction is required for anti-termination. We then applied single-particle cryo-EM analysis with rrnTAC assembled with and without S4 and gained a map at a global resolution of 3.3 Å. The structure reveals that rrnTAC adopts a similar strategy to λN-TAC in suppressing transcription termination and pausing. In vitro transcription assays and… Advisors/Committee Members: male (gender), Heyd, Florian (inspector), Freund, Christian (inspector), Said, Nelly (inspector), Wahl, Markus (firstReferee), Chakrabarti, Sutapa (furtherReferee).

Subjects/Keywords: antitermination; rRNA; SuhB; λN; Nus factors; co-transcriptional; ddc:572

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APA (6th Edition):

Huang, Y. (2021). Structure-based mechanisms of processive transcription anti-termination. (Thesis). Freie Universität Berlin. Retrieved from http://dx.doi.org/10.17169/refubium-27056

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Huang, Yong-Heng. “Structure-based mechanisms of processive transcription anti-termination.” 2021. Thesis, Freie Universität Berlin. Accessed January 16, 2021. http://dx.doi.org/10.17169/refubium-27056.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Huang, Yong-Heng. “Structure-based mechanisms of processive transcription anti-termination.” 2021. Web. 16 Jan 2021.

Vancouver:

Huang Y. Structure-based mechanisms of processive transcription anti-termination. [Internet] [Thesis]. Freie Universität Berlin; 2021. [cited 2021 Jan 16]. Available from: http://dx.doi.org/10.17169/refubium-27056.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Huang Y. Structure-based mechanisms of processive transcription anti-termination. [Thesis]. Freie Universität Berlin; 2021. Available from: http://dx.doi.org/10.17169/refubium-27056

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

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