Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A

Abstract

Mitochondria organize their genome in protein-DNA complexes called nucleoids. The mitochondrial transcription factor A (TFAM), a protein that regulates mitochondrial transcription, is abundant in these nucleoids. TFAM is believed to be essential for mitochondrial DNA compaction, yet the exact mechanism has not been resolved. Here we use a combination of single-molecule manipulation and fluorescence microscopy to show the nonspecific DNA-binding dynamics and compaction by TFAM. We observe that single TFAM proteins diffuse extensively over DNA (sliding) and, by collisions, form patches on DNA in a cooperative manner. Moreover, we demonstrate that TFAM induces compaction by changing the flexibility of the DNA, which can be explained by local denaturation of the DNA (melting). Both sliding of TFAM and DNA melting are also necessary characteristics for effective, specific transcription regulation by TFAM. This apparent connection between transcription and DNA organization clarifies how TFAM can accomplish two complementary roles in the mitochondrial nucleoid at the same time.

Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid

“…As the TFAM concentration increased, the binding events were not evenly distributed but accumulated in enlarged aggregates (Fig. 1B), consistent with the reported cooperative binding of TFAM to mtDNA (19). Although initial TFAM binding occurred as single events (Fig.…”

“…Rotary shadowing EM allowed us to see single TFAM molecules bound to mtDNA. Adding increasing amounts of TFAM to mtDNA did not result in a dispersed distribution of binding events but rather resulted in larger aggregates, consistent with cooperative binding (19). TFAM monomers are reported to slide along the DNA and are ∼100 times more likely to bind next to an already stationary TFAM patch than to naked DNA (19).…”

“…The ultrastructure of the mammalian nucleoid may be solely determined by binding of TFAM to mtDNA because TFAM is present at ∼1,000 copies per mtDNA molecule (6,11) and has structural (16)(17)(18) and biophysical properties (19,20,24,30) consistent with an important role in DNA compaction. However, this does not exclude that other proteins affect the packaging of mtDNA and determine nucleoid morphology (8).…”

“…Between the two HMG-box domains is a helical linker with a positively charged surface that interacts with the negatively charged backbone of the DNA (16,17). TFAM has the capacity to increase the flexibility of DNA to promote compaction (19,20). However, the mechanism of mtDNA compaction by TFAM is currently under debate.…”

“…can also bind to nonspecific DNA sequences in a cooperative manner to form stable protein patches (19). Beyond DNA bending, dimerization of TFAM has been reported to be necessary for full compaction of mtDNA into nucleoids (18,20).…”