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Structural and Mechanistic Studies of Ribonucleoprotein Particle Pseudouridine Synthase

Title: Structural and Mechanistic Studies of Ribonucleoprotein Particle Pseudouridine Synthase.
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Name(s): Zhou, Jing, author
Li, Hong, professor directing dissertation
Schlenoff, Joseph B., committee member
Striegel, Andre, committee member
Department of Chemistry and Biochemistry, degree granting department
Florida State University, degree granting institution
Type of Resource: text
Genre: Text
Issuance: monographic
Date Issued: 2010
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource
Language(s): English
Abstract/Description: Box H/ACA ribonucleoprotein particle (RNP), an RNA-guided RNA modification enzyme, is comprised of four conserved proteins (Cbf5, Nop10, Gar1 and L7Ae) and one guide RNA. This complex recognizes the substrates by the guide RNA and catalyzes the chemical modification by the protein partners. In archaea and eukaryotes, box H/ACA RNP is responsible for pseudouridylation in most stable RNAs, using more than 100 guide RNAs. In this thesis, we present the structural studies of this most complex pseudouridine synthase. Based on this complicated system, we extend our understanding of the catalytic mechanism of pseudouridylation. In Chapter 2, we present a crystal structure of the substrate binding archaeal box H/ACA RNP complex which shows the detailed information of the active site. The 5-fluorouridine on the target position of substrate RNA is fully docked and rearranged into (5S,6R)-5-fluoro-6-hydroxy-pseudouridine (f5oh6Ψ) similar to other known structures of non RNA-guide pseudouridine synthases. This structure also reveals the substrate RNA binding details and the functions of conserved protein partners. In Chapter 3, we apply the crystallization on various substrate analogs with the functional H/ACA RNP complex. We also combine the activity assays and structures, provide a complete view of the enzyme active site along the reaction pathway, and suggest pre-organization as an important catalytic strategy. In Chapter 4, we focus on the specificity of PUA domain of Cbf5 for the absolutely conserved ACA sequence on guide RNA. We discovered that the high specificity enables the Cbf5 to catch the short RNA substrate containing ACA at the 3'end during crystallization. The activity assay shows that ACA trinucleotides block the Cbf5 activity on the small hairpin RNA substrate in a non RNA-guide manner.
Identifier: FSU_migr_etd-7263 (IID)
Submitted Note: A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Degree Awarded: Summer Semester, 2010.
Date of Defense: April 20, 2010.
Bibliography Note: Includes bibliographical references.
Advisory Committee: Hong Li, Professor Directing Dissertation; Joseph B. Schlenoff, Committee Member; Andre Striegel, Committee Member.
Subject(s): Chemistry
Biochemistry
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_migr_etd-7263
Owner Institution: FSU

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Zhou, J. (2010). Structural and Mechanistic Studies of Ribonucleoprotein Particle Pseudouridine Synthase. Retrieved from http://purl.flvc.org/fsu/fd/FSU_migr_etd-7263