Fig. 2: [TFAM HMG-A and HMG-B stay bound to LSP DNA in the P-state]. | Nature Communications

Fig. 2: [TFAM HMG-A and HMG-B stay bound to LSP DNA in the P-state].

From: Sequence-specific dynamic DNA bending explains mitochondrial TFAM’s dual role in DNA packaging and transcription initiation

Fig. 2

A Schematic of PIFE assay to test whether TFAM HMG-box domains stay bound to LSP DNA (Model 1) or dissociate from the DNA in the P-State (Model 2). Two subclasses of Model 2 were tested against Model 1 by switching the dye positions on the DNA: (B–G) for testing Model 2A with Cy3 labeled at −12 end; and (H–M) for testing Model 2B with Cy3 labeled at −41 end. B Sample single-molecule PIFE (smPIFE) time traces of LSP and LSP + TFAM. C smFRET histograms for LSP (NTrace = 200), and LSP + TFAM (NTrace = 165). D Ensemble Cy3 fluorescence spectrum, showing a strong PIFE effect when TFAM C-tail mutant is added to LSP DNA labeled with Cy3 at −12 end. E Hybrid smFRET/smPIFE assay using LSP DNA labeled with Cy3 (donor) and Atto647N (acceptor) at −12 and −41 end, respectively. Correlative donor/acceptor fluorescence enhancement due to PIFE effect (i.e., an overall increase in fluorescence intensity) upon addition of TFAM is shown, particularly for the P-state, from single-molecule time traces and 2-D histograms of donor/acceptor fluorescence (bottom right: NTrace = 407 for LSP and 79 for LSP + TFAM). PIFE and anti-correlative donor/acceptor fluorescence change due to FRET were decoupled using the procedure and method described in Methods. An example of reconstructed smFRET and smPIFE time traces is shown for LSP alone (upper panel) or when TFAM is added (lower panel). The PIFE time traces represent all the emitted photon numbers normalized to LSP DNA alone case. F 2-D histogram plot of FRET-PIFE values for LSP alone (NTrace = 407) (left) and LSP + TFAM (NTrace = 79) (right). Time traces and 2-D histogram show strong PIFE signals when LSP-TFAM dwells in the low FRET state (i.e., P-state). G Ensemble fluorescence spectra of Cy3/Atto647N-labeled LSP DNA show a strong PIFE effect and a moderate FRET effect simultaneously occurring with the addition of TFAM C-tail mutant. H–M −41 end-labeled Cy3 counterpart of B–F. NTrace: 124 (LSP in I), 208 (LSP + TFAM in I), 417 (LSP in K and L), and 69 (LSP + TFAM in K and L).

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