Single molecule studies on the dynamics of the transcription initiation complex of yeast mitochondria

Abstract

The transcription initiation complex in the yeast mitochondria of Saccharomyces cerevisiae comprises the RNA polymerase, Rpo41, the initiation factor, Mtf1, and the DNA including 6 base pair promoter sequence. The Mtf1 is known to recognize and help to open the promoter region during the initiation stage, but its exact role and mechanism still remains unclear. We designed a multi-color single molecule FRET assay to directly measure the dynamics of the complex during transcription initiation. The labels on the DNA report on its opening-closing dynamics, while the label on Mtf1 report on the recruitment, dynamics, and dissociation of the initiation factor. From these measurements, we can correlate the promoter opening dynamics, factor binding/dissociation, and the transition to the elongation phase. Mtf1 is also associated with controlling the production of abortive RNA transcripts. We observed the scrunching motion during transcription by stepping along the DNA template with various combinations of nucleotide substrates. The FRET distribution shifted toward the high FRET region as we stepped further. From these observations, we propose a mechanistic model of the transcription initiation in the yeast mitochondria.