Consensus DNA sequence of human mitochondrial transcription factor A binding

Abstract

Human mitochondrial transcription factor A (h-mtTFA), a nucleus-encoded DNA binding protein, is the only accessory protein known to be required for accurate and efficient promoter recognition by mammalian mitochondrial RNA polymerase. Not only does this protein interact specifically with the upstream region of the promoters, the heavy-strand promoter (HSP) and the light-strand promoter (LSP), it also has high affinity for nonspecific DNA sequence. Cloning of the gene for h-mtTFA and nucleotide sequence analysis revealed that h-mtTFA is homologous to a group of nuclear high mobility group (HMG) proteins which have been implicated in diverse functions such as chromatin compaction and transcription stimulation. DNA binding sites for h-mtTFA have been deduced from the alignment with target sequences of related HMG proteins, methylation interference studies, and DNase I footprinting analysis. It remained unclear, however, whether this represents the only or the optimal binding sites for interaction with h-mtTFA. We have therefore utilized a SELEX (Systematic Evolution of Ligands by Exponential enrichment) analysis to define the nature of the h-mtTFA binding site, the range of DNA sequences recognized by the h-mtTFA proteins. There were two GTT (CTT) sites in several clones that have higher affinity. Furthermore, substitution of TT in GTT to CC or GG decreased the degrees of binding. These studies suggest that GTT is the consensus sequence as shown in sequence logos. LSP and HSP were more similar than other h-mtTFA binding regions to SELEX-generated sequence logos pattern.