Illuminating the DNA Binding Behavior of Mitochondrial Transcription Factor A

Abstract

Mitochondria are the energy producing organelles of eukaryotic cells. Owing to their endosymbiotic evolutionary history, they contain their own genome (mtDNA) that encodes for thirteen proteins essential for ATP production.In mammalian cells, multiple mtDNAs are compacted into protein-DNA complexes called “nucleoids”. A major component of these nucleoids is the mitochondrial transcription factor A (TFAM), a member of the high mobility group (HMG) family of proteins. This abundant protein binds DNA with little sequence specificity, and is able to coat the entire mtDNA molecule. It not only serves a role in mtDNA packaging, but is also required for mitochondrial transcription. At this point, dynamics of the TFAM-DNA interaction remain unclear.Experiments on single DNA molecules offer a very direct way to study TFAM dynamics. Tethered Particle Motion (TPM) experiments show that the system quickly equilibrates with the buffer, and that the end-to-end distance of the DNA decreases upon TFAM binding. Manipulations of single DNA molecules with two optical traps offer an explanation: TFAM decreases the DNA's stiffness (persistence length). A possible molecular mechanism for this decrease is that TFAM introduces bends in the DNA.Adding single-molecule fluorescence to the dual optical trap illuminates TFAM's binding behavior. Literally seeing TFAM on the DNA, we can derive on- and off-rates, and determine that TFAM does not bind cooperatively. Interestingly and seemingly unrelated to its role in DNA organization, we also observe that TFAM can rapidly bind single-stranded DNA (ssDNA), but not when the ssDNA is under tension. The physiological function of TFAM binding to ssDNA could be related to its regulation of transcription.