Mechanism and specificity of human mitochondrial transcription factor A binding to the light‐strand promoter

Several mitochondrial transcription factors and elements are required for transcription from the light‐strand promoter (LSP). These include mtTFA, mtTFB2, and mtRNAP. mtTFA is thought to interact with a binding site (BS) on LSP and recruit mtTFB2 and mtRNAP. It is not known exactly how the binding event occurs, or how the protein recognizes the binding site. The sequence of the putative BS for human, mouse, and bovine are rather disparate while the respective amino acid sequences indicate a high degree of similarity. This suggests that the binding of mtTFA is not directly related to the BS sequence. To investigate the binding mechanism, mtTFA from human, mouse, and bovine systems was assayed with a fluorescently‐labeled dsDNA containing the human mtTFA BS. The kinetics were studied through the use of fluorescence polarization. The K d for the interaction of mtTFA and the DNA binding site were found to be on the order of 1nM for the respective mtTFAs. Additionally, the stoichiometry of each DNA‐mtTFA interaction was determined to be two molecules of mtTFA for each binding site. Off‐rate measurements indicated that the human mtTFA‐BS dissociated over an order of magnitude more slowly than the corresponding complexes formed using the other TFAs. We predict a two‐step binding mechanism for formation of the mtTFA‐BS complex in which only cognate pairs are capable of transiting the second step efficiently.D N A + m t T F A ↔ I D N A ‐ m t T F A ↔ I ID N A x 002 A ; ‐ m t T F AThis work was supported by the American Recovery and Reinvestment Act (ARRA) Supplement to NIH/NIAID R01 AI053531 Picornavirus Genome Replication and the Paul Berg Endowment