| dc.identifier.uri | http://hdl.handle.net/11401/76940 | |
| dcterms.abstract | TFAM (mitochondrial transcription factor A) is a mitochondrial protein containing two tandem HMG (high-mobility group) boxes. It serves two essential functions in mitochondria: transcription activation and mtDNA (mitochondrial DNA) packaging. Interestingly, while its transcription activating function requires specific sequence recognition in order to specifically recognize the mitochondrial promoters, mtDNA packaging occurs mainly through non-sequence specific binding. However, the mechanism of nucleic acid recognition for both specific and nonspecific sequences has not yet been established despite the determination of TFAM crystal structures in complex with promoter sequences. The structures reveal that most of the interactions between TFAM and the promoters are non-sequence specific. However, I found two sequence specific interactions with two guanines separated by 10 variable nucleotides (GN10G). These guanines hydrogen-bond to two TFAM residues in the HMG box 1 and 2. Intriguingly, these interactions are conserved in all TFAM structures with both promoter and nonspecific sequences, indicating that TFAM might recognize this GN10G consensus regardless of substrate specificity. Here, I present a crystal structure of TFAM in complex with a non-specific sequence (NS2) containing a GN10G consensus. The structure reveals consistent binding through the consensus, leading to the hypothesis that TFAM prefers to recognize the GN10G consensus on DNA substrates for its variable functions. In vitro transcription assays show that the GN10G consensus plays an important role in mitochondrial transcription, and EMSA (electrophoretic mobility shift assay) and other binding assays reveal that a GN10G consensus contributes to directing TFAM binding, indicating that it appears to have a role in mtDNA packaging. In addition, the TFAM-NS2 structure displays a unique interaction to a DNA nick composed of two DNA ends, which is consistent with previous studies about the preferential binding of TFAM to DNA damaged regions. End-joining assays suggest that TFAM is able to facilitate DNA ligation events, implicating its possible role in double-strand break repair in mitochondria. These studies will shed light not only on the mechanism of TFAM binding, but also on the involvement of TFAM in mitochondrial DNA repair. | |
| dcterms.available | 2017-09-20T16:51:29Z | |
| dcterms.contributor | Advisor: Garcia-diaz, Miguel | |
| dcterms.contributor | Committee members: Garcia-Diaz, Miguel; Glynn, Steven; Hollingsworth, Nancy; Schaerer, Orlando; Bogenhagen, Daniel | |
| dcterms.creator | Choi, Woo Suk | |
| dcterms.date | 2016 | |
| dcterms.dateAccepted | 2017-09-20T16:51:29Z | |
| dcterms.description | Dissertation | |
| dcterms.description | Department of Biochemistry and Structural Biology | |
| dcterms.extent | 89 pages | |
| dcterms.format | application/pdf | |
| dcterms.issued | 2016-08 | |
| dcterms.language | en | |
| dcterms.provenance | Made available in DSpace on 2017-09-20T16:51:29Z (GMT). No. of bitstreams: 1
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Previous issue date: 42370 | |
| dcterms.provenance | Item withdrawn by Jason Torre (fjason.torre@stonybrook.edu) on 2018-01-09T22:21:34Z
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Choi_grad.sunysb_0771E_13020.pdf: 3029206 bytes, checksum: 287f82a72a953da81fc74898b8223fdc (MD5) | |
| dcterms.provenance | Item reinstated by Jason Torre (fjason.torre@stonybrook.edu) on 2019-11-25T17:22:58Z
Item was in collections:
Stony Brook Theses and Dissertations Collection (ID: 627)
No. of bitstreams: 2
Choi_grad.sunysb_0771E_13020.pdf.txt: 156313 bytes, checksum: 4ebad0307f0316be2333ee187fb4dea8 (MD5)
Choi_grad.sunysb_0771E_13020.pdf: 3029206 bytes, checksum: 287f82a72a953da81fc74898b8223fdc (MD5) | |
| dcterms.provenance | Item reinstated by Jason Torre (fjason.torre@stonybrook.edu) on 2019-11-25T17:23:00Z
Item was in collections:
Stony Brook Theses and Dissertations Collection (ID: 627)
No. of bitstreams: 2
Choi_grad.sunysb_0771E_13020.pdf.txt: 156313 bytes, checksum: 4ebad0307f0316be2333ee187fb4dea8 (MD5)
Choi_grad.sunysb_0771E_13020.pdf: 3029206 bytes, checksum: 287f82a72a953da81fc74898b8223fdc (MD5) | |
| dcterms.publisher | Stony Brook University | |
| dcterms.subject | DNA repair, Double-strand break, GN10G, mitochondrial transcription, mtDNA packaging, TFAM | |
| dcterms.title | Structural and Biochemical Analysis of Substrate Recognition by Mitochondrial Transcription Factor A (TFAM) | |
| dcterms.type | Text | |
