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Bachelor of Science, 2004
Samara State University
Thesis Advisor: Dmitry Temiakov, Ph.D.
Cell and Molecular Biology Program
Science Center, Room 290
Thursday, April 15, 2010
Mitochondria play an important role in a number of cellular processes such as apoptosis, heme biosynthesis and aging. Mitochondria maintain their own genome (mtDNA), which encodes several proteins required for oxidative phosphorylation. MtDNA was shown to contain two major promoters: LSP and HSP1. Expression of human mitochondrial genes is carried by single-subunit T7 phage-like RNA polymerase (mtRNAP) that requires at least two transcription initiation factors: TFA and TFB2.
We reconstituted human mitochondrial in vitro transcription system that encompasses highly purified human mitochondrial RNA polymerase as well as TFA and TFB2. We found that TFA and TFB2 act synergistically and increase transcription efficiency 20- to 200-fold compared with RNAP alone or when only one of these two factors was present. Using in vitro transcription assays we established that MRPL12 and TFB1, previously thought to modulate mitochondrial gene expression, do not affect in vitro transcription by mtRNAP. Our transcription assays also demonstrated that putative HSP2 promoter cannot be transcribed in vitro; raising the possibility that it may be not a functional promoter. The results of our study indicate that transcription of human mitochondrial genes requires two promoters (LSP and HSP1) and only two essential initiation factors - TFA and TFB2.
In vitro transcription assays demonstrated that TFB2 facilitates promoter melting but may not be essential for promoter recognition. Additionally, cross-linking experiments revealed that TFB2 directly contacts promoter DNA near the transcription start site and interacts with the priming NTP, suggesting that it acts as a transient component of the catalytic center of the initiation complex. Protein mapping indicated that a region of N-terminal domain of TFB2 is involved in simultaneous interactions with the priming substrate and the templating (+1) DNA base, assuring correct positioning of the priming NTP during transcription initiation.
Our data indicate that the transcriptional machinery in human mitochondria has evolved into a system that combines features inherited from self-sufficient, T7-like RNA polymerase and those typically found in systems comprising cellular multi-subunit polymerases, and provide insights into the molecular mechanisms of transcription.