Dissertation/Thesis Abstract

Structural and Functional Studies of E. coli Conjugative Relaxase-Helicase TraI and Arabidopsis thaliana Protein Arginine Methyltransferase 10 (PRMT10)
by Cheng, Yuan, Ph.D., The University of North Carolina at Chapel Hill, 2011, 163; 3456259
Abstract (Summary)

TraI, a bifunctional enzyme containing relaxase and helicase activities, initiates and drives the conjugative transfer of the E. coli F plasmid. In this work, we investigated the function of the putative RecD-like domain of TraI (284-821). We established that TraI 284-821 can bind nucleotides, but only retained residual ATPase activity likely due to the loss of critical catalytic motifs. We also examined the DNA binding properties of the TraI helicase. We showed that TraI binds to single-stranded DNA (ssDNA) with a site-size of approximately 25 nucleotides and low cooperativity. A double-stranded DNA (dsDNA) binding site was identified within the N-terminal region of TraI (1-858), outside the core helicase motifs of TraI. We further characterized the impacts of ionic strength, nucleotide binding and base composition on TraI-DNA interaction. Finally, we elucidated the solution structure of TraI using small-angle x-ray scattering. Taken together, these data resulted in the assembly of a model for TraI’s multi-domain helicase activity.

Protein arginine methyltransferase 10 (PRMT10) regulates flowering-time in Arabidposis thaliana. Here, we present the 2.6 Å resolution crystal structure of PRMT10 that reveals significant structural features unique to PRMT10, including a long dimerization arm and distinct accessibility to the active site. Our data also showed that the N-terminal thirty residues of PRMT10 impact substrate specificity, and that PRMT10 activity was dependent on the sequences distal from the substrate methylation site. We further established that PRMT10 dimerization is required for activity and used structure-based molecular dynamics to indicate how dimerization affects functionally-essential PRMT10 domain motions. Taken together, our results provide substantial insights into the mechanism governing the unique enzymatic function and substrate specificity of PRMT10.

Indexing (document details)
Advisor: Redinbo, Matthew R., Kuhlman, Brian
Commitee: Dohlman, Henrick, Slep, Kevin, Thomas, Christopher, Wolfgang, Matthew
School: The University of North Carolina at Chapel Hill
Department: Biochemistry & Biophysics
School Location: United States -- North Carolina
Source: DAI-B 72/08, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Biochemistry, Biophysics
Keywords: Arabidopsis thaliana, Arginine, Helicase, Methyltransferase, Relaxase, TraI
Publication Number: 3456259
ISBN: 978-1-124-65602-1
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