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Structural and functional characterization of Rtf1 and insight into its role in transcriptional regulation

Wier, Adam D. (2015) Structural and functional characterization of Rtf1 and insight into its role in transcriptional regulation. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Originally discovered in a search for RNA polymerase II-associated factors, the Paf1 complex (Paf1C) is best characterized for its roles in regulating transcription elongation. The complex co-localizes with RNA polymerase II from the promoter to the 3’ end of genes and has been linked to a growing list of transcription-related processes including: elongation through chromatin, histone modifications, and recruitment of factors important in transcript maturation. The complex is conserved throughout eukaryotes and is comprised of the proteins Paf1, Ctr9, Cdc73, Rtf1, and Leo1. The domain structures of Paf1C subunits are largely undefined and have few clear homologs, making it difficult to postulate for or localize functions to the individual subunits. To understand mechanistically how Paf1C coordinates its functions and interactions, I took an approach utilizing biochemical, biophysical, and structural techniques to characterize proteins within Paf1C, specifically focusing on the Rtf1 subunit.
The goal of my thesis work was to determine the molecular mechanism by which Rtf1 influences transcription and chromatin structure. To this end I focused on studying different functional domains within Rtf1. I provided a molecular description of how Rtf1 mediates Paf1C recruitment to elongating RNA polymerase II. Recruitment of Rtf1 is controlled by its centrally located Plus3 domain and a direct interaction with the conserved elongation factor Spt5. I solved the co-crystal structure of the human Plus3 domain bound to a phosphorylated C-terminal repeat of Spt5. The structure revealed the basis for recognition of the repeat motif of Spt5, an important component in the recruitment of regulatory factors to RNA polymerase II. I have performed further structural characterization of Rtf1, studying the N-terminal histone modification domain. To gain insight into the molecular mechanism underlying the domain’s function, I successfully crystallized and solved the structure of a minimal region of Rtf1 that is necessary and sufficient for Rtf1-mediated histone modifications. The structure, paired with conservation analysis and genetic phenotype data, have allowed us to identify important surfaces on Rtf1 that function in regulating chromatin structure. Taken together these studies shed new light onto the mechanism by which Paf1C influences the complex network of regulatory interactions required for eukaryotic transcription.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Wier, Adam D.adw46@pitt.eduADW46
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairVanDemark, Andrew P.andyv@pitt.eduANDYV
Committee MemberPipas, James Mpipas@pitt.eduPIPAS
Committee MemberArndt, Karen M.arndt@pitt.eduARNDT
Committee MemberRosenberg, John M.jmr@pitt.eduJMR
Committee MemberSchmidt, Martin C.mcs2@pitt.eduMCS2
Date: 14 January 2015
Date Type: Publication
Defense Date: 20 August 2014
Approval Date: 14 January 2015
Submission Date: 29 August 2014
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 197
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: X-ray crystallography, Paf1 complex, Transcription, RNA Polymerase II
Date Deposited: 14 Jan 2015 17:07
Last Modified: 14 Jan 2020 06:15


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