Báo cáo khoa học: Transcription termination at the mouse mitochondrial H-strand promoter distal site requires an A/T rich sequence motif and sequence specific DNA binding proteins

Transcription Termination at the Mouse Mitochondrial H-Strand Promoter Distal Site Requires an A/T Rich Sequence Motif and Sequence Specific DNA Binding Proteins

Authors: Vijayasarathy Camasamudram, Ji-Kang Fang, Narayan G. Avadhani

Field: Biochemistry, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Document Content: This study investigates the termination of mitochondrial H-strand transcription in mammalian cells, specifically focusing on the promoter distal site. The research characterizes a specific DNA sequence motif, termed D-TERM, located upstream of the tRNA^Phe gene, which is critical for transcription termination. Gel-shift analysis revealed that this D-TERM sequence forms complexes with proteins found in mouse liver mitochondrial extracts in a sequence-specific manner. Further purification identified two major proteins, 45 kDa and 70 kDa, involved in this interaction. The D-TERM DNA was demonstrated to mediate transcription termination in a unidirectional manner in an in vitro system when these purified proteins were present. This work identifies a novel mitochondrial transcription termination system with characteristics similar to those observed in sea urchins and nuclear RNA Pol I and Pol II termination systems.

Detailed Table of Contents:

• Introduction
• Materials and Methods
• Preparation of mt extract capable of transcription initiation
• Preparation of mt extract for DNA binding
• Partial purification of mt transcription termination factor by DNA affinity chromatography
• Gel mobility shift assays
• DNase I footprint analysis
• Methylation interference analysis
• UV induced protein-DNA cross-linking
• South-western blotting
• Construction of transcription vectors and assay of transcription termination
• Results
• Locations and sequence properties of the proximal and distal transcription terminators
• Protein binding property of the distal terminator sequence
• Nature of mt proteins binding to the D-TERM DNA sequence
• Purification of D-TERM binding proteins
• Factor-dependent transcription termination by D-TERM DNA under in vitro conditions
• Discussion
• Acknowledgements
• References