The Biophysical and Biochemical Characterization of the Transcription of Mitochondrial DNA
Open Access
- Author:
- Oliver, Gwen
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Craig Cameron, Thesis Supervisor
Joseph C. Reese, Thesis Honors Advisor - Keywords:
- transcription
TFAM
mitochondria
mtDNA - Abstract:
- Mitochondrial DNA (mtDNA) is critical to the health of all individuals, but little is known about its regulation. In an effort to elucidate its mechanisms of regulation further, we set two goals to study the transcription of mitochondrial DNA: First, to develop a method to visualize mtDNA using atomic force microscopy and second, to characterize the effect of a mutant of mitochondrial transcription factor A (TFAM P178L) on transcription of mtDNA. This mutant has been linked to a fatal phenotype in homozygous individuals and we suspect it could be related to dysregulation of the transcription of mtDNA. To develop a method to visualize mtDNA using atomic force microscopy (AFM), we determined the appropriate mtDNA templates for visualization and the optimal DNA fixation technique. Our results show that authentic mtDNA templates are more robust than chimeric templates, and that nickel-mediated deposition is preferable to APTES-mediated deposition. To characterize the effect of TFAM P178L on mitochondrial transcription, we first used transcription assays. TFAM P178L exhibited unique activity in transcription assays: there was reduced repression at higher concentrations of TFAM P178L from the light strand promoter and reduced activation at lower concentrations from the heavy strand promoter 1 compared to TFAM wild type. We then used AFM assays to observe mtDNA conformation. In these assays, we observed delocalized looping and lower compaction in the presence of the mutant. Surprisingly, DNA binding affinity assays showed that TFAM P178L has a higher binding affinity to mtDNA compared to TFAM wild type. These differences in structure and binding give us a broader picture of the mutant phenotype and are likely linked to the observed changes in transcriptional output and may contribute to the lethal phenotype in humans homozygous for the mutant allele.