Human mitochondrial transcription factor A and promoter spacing integrity are required for transcription initiation.
The two major promoters for transcription of the human mitochondrial genome are located near each other in the displacement-loop region of the molecule. Previous work has localized these promoters to regions of < 100 nucleotides each; the DNA sequence at the transcription start site is stringently required, as is the region from -10 to -40 base pairs upstream of each respective start site. Each upstream site is recognized and bound by human mitochondrial transcription factor A (h-mtTFA), an event previously shown to be important for transcriptional activation. We report here results using recombinant h-mtTFA that demonstrate the dependence of transcription initiation of h-mtTFA. In addition, altering the distance between the h-mtTFA binding site and the transcription start site greatly impairs transcription initiation efficiency. The decrease in transcription initiation efficiency was shown to be a consequence of altering the position of h-mtTFA binding as opposed to the strength of h-mtTFA binding, as judged by DNA footprinting ability. Analysis of a chimeric yeast-human promoter revealed that the yeast mtTFA homologue cannot substitute for the human protein, even when bound at an appropriate position upstream of the human transcription start site.
Dairaghi
D J
DJ
Department of Developmental Biology, Stanford University School of Medicine, CA 94305-5427, USA.
Shadel
G S
GS
Clayton
D A
DA
D001482:Base Composition; D001483:Base Sequence; D002678:Chimera; D003001:Cloning, Molecular; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000096:biosynthesis* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007624:KB Cells; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013698:Templates, Genetic; D014157:Transcription Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C499631:TFAM protein, human; D014157:Transcription Factors; C422562:mitochondrial transcription factor A; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I