MECHANISM AND SPECIFICITY OF MAMMALIAN MITOCHONDRIAL TRANSCRIPTION FACTORS A BINDING TO THE LIGHT-STRAND PROMOTER OF HUMAN MITOCHONDRIAL DNA
Open Access
- Author:
- Kumar, Nitin
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Craig Eugene Cameron, Thesis Supervisor
Craig Eugene Cameron, Thesis Supervisor
David Scott Gilmour, Faculty Reader
Craig Eugene Cameron, Thesis Honors Advisor - Keywords:
- mitochondrial transcription
transcription factor A
light-strand promoter - Abstract:
- The mitochondrion is well known as the “power house” of the cell, in that it is responsible for the majority of energy production within the cell, in the form of ATP. The human mitochondrion possesses its own genome, which consists of a 16,569 bp circular DNA that encodes 13 subunits of the electron transport chain, as well as 22 tRNAs and 2 rRNAs. Transcription of these genes is started from one of three promoters: lsp, hsp1, or hsp2 (Gangelhoff, Mungalachetty et al. 2009). Mitochondrial transcription is, thus far, not entirely understood. As in nuclear transcription, there are several important nuclear-encoded proteins involved (Shutt, Lodeiro et al. 2010). These include mtTFA, mtTFB2, and mtRNAP. A combination of these proteins is known to be necessary for effective transcription. This study focuses on the interaction of mtTFA with its putative binding site on the light-strand promoter. mtTFA is thought to interact with the DNA and recruit mtTFB2 and mtRNAP to this promoter. However, it is not known exactly how the binding event occurs, or how the protein recognizes the binding site. Furthermore, the sequence of the putative binding sites for human and mouse, and bovine systems are rather disparate. The sequences of each of the proteins are nearly identical, and based on modeling data, the structures are also thought to be very similar. This information indicates that the binding of mtTFA is not directly related to the sequence of the binding site. Perhaps there is a two step process with an initial binding followed by a second step with more specific requirements that involves a bending or unwinding of the DNA. In order to investigate the mtTFA binding mechanism, mtTFA from human, mouse, and bovine systems were assayed with a double-stranded, fluorescently-labeled oligonucleotide containing the human mtTFA binding site sequence. The kinetics of this interaction were studied through the use of fluorescence anisotropy at equilibrium and at several shorter time scales to learn about the equilibrium dissociation constant, and on- and off-rates.