In organello footprint analysis of human mitochondrial DNA: human mitochondrial transcription factor A interactions at the origin of replication.
Using in organello footprint analysis, we demonstrate that within human placental mitochondria there is a high level of protein-DNA binding at regularly phased intervals throughout a 500-bp region encompassing the D-loop DNA origins and two promoter regions. Comparison with in vitro DNase I protection studies indicates that this protein-DNA interaction is due to non-sequence-specific binding by human mitochondrial transcription factor A (h-mtTFA). Since h-mtTFA can bend and wrap DNA, like its yeast counterpart ABF2, a primary function of h-mtTFA appears to be specific packaging of the mitochondrial DNA control region in vivo. Intervals of protein binding coincide with the spacing of the RNA start sites and prominent D-loop DNA 5' ends, suggesting a role for phased h-mtTFA binding in defining transcription and H-strand DNA replication origins. Significant protein-DNA interaction was also observed within the human homolog of conserved sequence block 1, both in organello and in vitro, using purified h-mtTFA.
Ghivizzani
S C
SC
Department of Immunology and Medical Microbiology, University of Florida, College of Medicine, Gainesville 32610-0266.
Madsen
C S
CS
Nelen
M R
MR
Ammini
C V
CV
Hauswirth
W W
WW
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics*; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D008928:Mitochondria / Q000502:physiology*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D029867:Xenopus Proteins
D017931:DNA Primers; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; C499631:TFAM protein, human; D015534:Trans-Activators; D014157:Transcription Factors; C097078:XL-MTTFA protein, Xenopus; D029867:Xenopus Proteins; C422562:mitochondrial transcription factor A