Regulatory region of the human mitochondrial transcription termination factor gene

Abstract

To understand the regulation of the human mTERF gene, the 5``-flanking region was characterized. Sequence analysis revealed that it contains three exons (-1274~+37, +580~+639 and +5938~+7794) and two introns (+37~+580 and +639~+5938). This 5?region contained putative binding sites for various transcription factors. Instead of TATA or CAAT box, there were binding sites of NRF-2 factors near the transcription start site (+1). For the purpose of defining sequences that regulate expression of the mTERF, 5``-deletion mutants were cloned to the upstream region of CAT gene in promoter-less pCAT3-Basic vector. The transcriptional activities of these 5``-deletion mutants were analyzed with CAT activity after transfection into HeLa cells. Analysis of deletion mutants revealed four regions, that could contain a positive (-1274~-949), negative (-254~-59) regulatory elements, and two NRF-2 binding sites (-57 and -38), respectively. These results suggest the presence of trans-acting factors that act on these positive and negative regulatory elements and regulate the transcription of the mTERF gene. Other results confirmed that two binding sites of NRF-2 were important in the transcription of the mTERF gene. Gel mobility shift assays were performed to prove the presence of specific factors binding to these sites. The fact that GG bases, of the NRF-2 consensus sequence GGAA, determined the specificity of one band, was found by competition experiments. These results confirmed that it was made by specific interactions between some proteins and probes. From these results, the possibility that the protein interacting probes might be NRF-2 factor was very high. If so, it would provide another link between nuclear and mitochondrial gene expression regulated by not NRF-1 but NRF-2 as a major factor.