Yeast mitochondrial (mt) RNA polymerase (RNAP) is an intriguing enzyme in that, while the polymerase Rpo41 is homologous to the single-subunit T7/T3 RNAP, its transcription factor Mtf1 appears to function similarly to the initiation factors of multi-subunit RNAPs. Yet, Mtf1's primary structure does not bear any similarity to Eukaryotic transcription factors. Fluorescence quenching measurement of each base (1) and DNA-protein cross-linking study (2) have demonstrated that Mtf1 facilitates the promoter melting and traps the non-template strand by direct interaction with the DNA. However, the dynamics of polymerase/factor binding and promoter melting are not well understood. Using single molecule techniques, we found that the complex of template DNA with Rpo41/Mtf1 undergoes opening-closing transitions without the proteins leaving the complex. Adding ribonucleotides decreased the closing rate for the cognate nucleotide but not for non-cognate nucleotides, implying that only the cognate nucleotide can stay long enough in the pre-initiation complex and stabilize the complex for accurate transcription. Rpo41 alone without Mtf1 can form the open complex on mismatched pre-melted template and Mtf1 further stabilizes this at open state by ∼50-fold. Single molecule studies provide insights into the mechanism of promoter recognition and opening by cooperative action of the polymerase and the factor.(1) G. Tang, S. Paratkar, and S. patel, “Fluorescence Mapping of the Open Complex of Yeast Mitochondrial RNA Polymerase”, J. Biol. Chem. 284, 5514 (2009)(2) S. Paratkar and S. patel, “Mitochondrial Transcription Factor Mtf1 Traps the Unwound Non-template Strand to Facilitate Open Complex Formation”, J. Biol. Chem. 285, 3949 (2010)