A model for transcription initiation in human mitochondria.
Bottom Line: In this study we mapped the binding sites of the core transcription initiation factors TFAM and TFB2M on human mitochondrial RNA polymerase, and interactions of the latter with promoter DNA.This allowed us to construct a detailed structural model, which displays a remarkable level of interaction between the components of the initiation complex (IC).The architecture of the mitochondrial IC suggests mechanisms of promoter binding and recognition that are distinct from the mechanisms found in RNAPs operating in all domains of life, and illuminates strategies of transcription regulation developed at the very early stages of evolution of gene expression.
Affiliation: Department of Cell Biology, School of Osteopathic Medicine, Rowan University, 2 Medical Center Dr., Stratford, NJ 08084, USA.Show MeSH
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Mentions: The remnants of the ancient single-subunit RNA polymerases of the Pol A family can be found in modern bacteriophages and DNA-maintaining organelles, such as plastids and mitochondria. In the latter, these RNAPs are charged with responsibility to synthesize mRNA, tRNA and rRNA, and to make RNA primers for replication (1–3). Even though some mitochondrial genomes are small (e.g. human mtDNA), regulation of mitochondrial gene expression is an elaborate process that occurs at various stages and involves many auxiliary factors and DNA and RNA modifying enzymes (4–8). Numerous mitochondrial dysfunctions are associated with defects in expression of mitochondrial genes and contribute to aging and severe pathologies and dysfunctions (9). At the beginning of the gene expression process, transcription of human mitochondrial DNA requires assembly of an initiation complex (IC) composed of mitochondrial RNA polymerase (mtRNAP) and two core transcription factors: TFAM and TFB2M (10–12). Recent studies demonstrated that mtRNAP is recruited to the promoter by formation of direct interactions with the nucleoid protein, TFAM (Figure 1A) (13,14). The resulting complex, called the pre-IC, lacks specificity toward DNA and cannot initiate transcription unless another transcription factor, TFB2M, is bound (13). Upon binding, the N-terminus of TFB2M reaches the active site of mtRNAP where it interacts with the priming substrate and assists in promoter melting (15). However, neither TFB2M nor TFAM binding sites on mtRNAP have been identified and thus the overall architecture of the IC as well as the pre-IC remains obscure. In this work, using biochemical, genetic and structural data we build a comprehensive map of interactions between all components of the IC. These data allowed us to construct a model of transcription initiation which is essential for understanding of molecular mechanisms of promoter binding and recognition and future studies of regulation of gene expression in human mitochondria.
Affiliation: Department of Cell Biology, School of Osteopathic Medicine, Rowan University, 2 Medical Center Dr., Stratford, NJ 08084, USA.