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Termination of mitochondrial (mt) H-strand transcription in mammalian cells occurs at two distinct sites on the genome. The first site of termination, referred to as mt-TERM occurs beyond the 16 S rRNA gene. However, the second and final site of termination beyond the tRNA Thr gene remains unclear. In this study we have characterized the site of termination of the polycistronic distal gene transcript beyond the D-loop region, immedi-ately upstream of the tRNA Phe gene. | Eur. J. Biochem. 270 1128-1140 2003 FEBS 2003 doi 10.1046 j.1432-1033.2003.03461.x Transcription termination at the mouse mitochondrial H-strand promoter distal site requires an A T rich sequence motif and sequence specific DNA binding proteins Vijayasarathy Camasamudram Ji-Kang Fang and Narayan G. Avadhani Laboratories of Biochemistry Department of Animal Biology School of Veterinary Medicine University of Pennsylvania Philadelphia Pennsylvania USA Termination of mitochondrial mt H-strand transcription in mammalian cells occurs at two distinct sites on the genome. The first site of termination referred to as mt-TERM occurs beyond the 16 S rRNA gene. However the second and final site of termination beyond the tRNAThr gene remains unclear. In this study we have characterized the site of termination of the polycistronic distal gene transcript beyond the D-loop region immediately upstream of the tRNAPhe gene. This region termed D-TERM maps to nucleotides 16274-16295 of the mouse genome and includes a conserved A T rich sequence motif AATAAA as a part of the terminator. Gel-shift analysis showed that the 22 bp D-TERM DNA forms two major complexes with mouse liver mt extract in a sequence-specific manner. Protein purification by DNA-affinity chromatography yielded two major proteins of 45 kDa and 70 kDa. Finally the D-TERM DNA can mediate transcription termination in a unidirectional manner in a HeLa mt transcription system only in the presence of purified mouse liver mt D-TERM DNA binding proteins. We have therefore characterized a novel mt transcription termination system similar in some properties to that of sea urchin as well as the nuclear RNA Pol I and Pol II transcription termination systems. Keywords mitochondria transcription termination mitochondrial H-strand transcription D-loop binding proteins polyadenylation. The mitochondrial mt genome in mammalian cells is a double-stranded circular DNA which encodes two rRNAs 22 tRNAs D-loop primer RNAs for DNA .
