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.
|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|
|Keywords:||Arabidopsis thaliana, Arginine, Helicase, Methyltransferase, Relaxase, TraI|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be