The dynamic landscape of transcription initiation in yeast mitochondria.

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA-DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA-DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.

Sequence-dependent DNA condensation as a driving force of DNA phase separation.

The physical properties of DNA have been suggested to play a central role in spatio-temporal organization of eukaryotic chromosomes. Experimental correlations have been established between the local nucleotide content of DNA and the frequency of inter- and intra-chromosomal contacts but the underlying physical mechanism remains unknown. Here, we combine fluorescence resonance energy transfer (FRET) measurements, precipitation assays, and molecular dynamics simulations to characterize the effect of DNA nucleotide content, sequence, and methylation on inter-DNA association and its correlation with DNA looping. First, we show that the strength of DNA condensation mediated by poly-lysine peptides as a reduced model of histone tails depends on the DNA's global nucleotide content but also on the local nucleotide sequence, which turns out to be qualitatively same as the condensation by spermine. Next, we show that the presence and spatial arrangement of C5 methyl groups determines the strength of inter-DNA attraction, partially explaining why RNA resists condensation. Interestingly, multi-color single molecule FRET measurements reveal strong anti-correlation between DNA looping and DNA-DNA association, suggesting that a common biophysical mechanism underlies them. We propose that the differential affinity between DNA regions of varying sequence pattern may drive the phase separation of chromatin into chromosomal subdomains.