Relative abundance of the human mitochondrial transcription system and distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression.
Human mitochondrial transcription requires the bacteriophage-related RNA polymerase, POLRMT, the mtDNA-binding protein, h-mtTFA/TFAM, and two transcription factors/rRNA methyltransferases, h-mtTFB1 and h-mtTFB2. Here, we determined the steady-state levels of these core transcription components and examined the consequences of purposeful elevation of h-mtTFB1 or h-mtTFB2 in HeLa cells. On a per molecule basis, we find an approximately 6-fold excess of POLRMT to mtDNA and approximately 3-fold more h-mtTFB2 than h-mtTFB1. We also estimate h-mtTFA at approximately 50 molecules/mtDNA, a ratio predicted to support robust transcription, but not to coat mtDNA. Consistent with a role for h-mtTFB2 in transcription and transcription-primed replication, increased mitochondrial DNA and transcripts result from its over-expression. This is accompanied by increased translation rates of most, but not all mtDNA-encoded proteins. Over-expression of h-mtTFB1 did not significantly influence these parameters, but did result in increased mitochondrial biogenesis. Furthermore, h-mtTFB1 mRNA and protein are elevated in response to h-mtTFB2 over-expression, suggesting the existence of a retrograde signal to the nucleus to coordinately regulate expression of these related factors. Altogether, our results provide a framework for understanding the regulation of human mitochondrial transcription in vivo and define distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression that together likely fine-tune mitochondrial function.
Cotney
Justin
J
Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, 440 Clifton Road N.E., Atlanta, Georgia 30322, USA.
Wang
Zhibo
Z
Shadel
Gerald S
GS
D000617:Aminoglycosides / Q000494:pharmacology; D004272:DNA, Mitochondrial / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005786:Gene Expression Regulation; D050259:Genes, Mitochondrial; D006367:HeLa Cells; D006801:Humans; D008564:Membrane Potentials; D008780:Methyltransferases / Q000378:metabolism / Q000502:physiology*; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010085:Oxidative Phosphorylation; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D021381:Protein Transport; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic
D000617:Aminoglycosides; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D021122:Protein Subunits; C499632:TFB1M protein, human; D014157:Transcription Factors; D008780:Methyltransferases; C462443:TFB2M protein, human; D012321:DNA-Directed RNA Polymerases; C519567:POLRMT protein, human; C100305:kasugamycin