Characterization of the 5′-flanking region of the rat gene for mitochondrial transcription factor A (Tfam)

Abstract

Mitochondrial transcription factor A (Tfam) is essential for the initiation of transcription and the replication of mitochondrial DNA (mtDNA). The 5′-upstream region of the rat Tfam gene was isolated from rat genomic DNA by extending the 5′ sequence of newly identified Tfam cDNA using PCR-based Genome Walker. The identified rat Tfam gene showed little sequence homology with the upstream region of human Tfam. No typical TATA or potential nuclear respiratory factor 1 (NRF-1) binding site was found, whereas three potential binding sites for Sp1 and one for NRF-2 were present in the proximal upstream region. Transfection of the Tfam 5′-upstream region ligated to luciferase into rat skeletal muscle L6 cells demonstrated that the promoter activity was 10 times higher than that of control vectors. Despite the absence of the NRF-1 consensus binding sequence, co-transfection of human NRF-1 expression vector increased the Tfam promoter activity 2-fold. The gel mobility shift assays for NRF-1 demonstrated that NRF-1 could bind to the proximal region of the promoter between −112 and +49. The addition of 50 mM glucose for 24 h increased Tfam promoter activity by up to 2-fold, but 100 μM H 2O 2 for 24 h repressed the activity to 40% of the control. From these results, the sequence presented here is that of authentic rat Tfam promoter and it is hoped that it will prove useful in studies of mtDNA transcription and replication.