非表示:
キーワード:
Animals
Base Sequence
Binding Sites/genetics
DNA Topoisomerases, Type I/metabolism
DNA, Mitochondrial/chemistry/genetics/metabolism
DNA, Superhelical/chemistry/genetics/metabolism
DNA-Binding Proteins/*genetics/metabolism
Humans
Immunoblotting
Mitochondria/*genetics/metabolism
Mitochondrial Proteins/*genetics/metabolism
Molecular Sequence Data
Mutation
Promoter Regions, Genetic/genetics
Protein Binding/drug effects
Sf9 Cells
Sodium Chloride/pharmacology
Spectrophotometry
Spodoptera
Transcription Factors/*genetics/metabolism
*Transcription, Genetic
要旨:
Transcription factor A (TFAM) functions as a DNA packaging factor in mammalian mitochondria. TFAM also binds sequence-specifically to sites immediately upstream of mitochondrial promoters, but there are conflicting data regarding its role as a core component of the mitochondrial transcription machinery. We here demonstrate that TFAM is required for transcription in mitochondrial extracts as well as in a reconstituted in vitro transcription system. The absolute requirement of TFAM can be relaxed by conditions that allow DNA breathing, i.e., low salt concentrations or negatively supercoiled DNA templates. The situation is thus very similar to that described in nuclear RNA polymerase II-dependent transcription, in which the free energy of supercoiling can circumvent the need for a subset of basal transcription factors at specific promoters. In agreement with these observations, we demonstrate that TFAM has the capacity to induce negative supercoils in DNA, and, using the recently developed nucleobase analog FRET-pair tC(O)-tC(nitro), we find that TFAM distorts significantly the DNA structure. Our findings differ from recent observations reporting that TFAM is not a core component of the mitochondrial transcription machinery. Instead, our findings support a model in which TFAM is absolutely required to recruit the transcription machinery during initiation of transcription.
