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Characterization of transcription termination factors in mammalian mitochondria


Transcription from the mitochondrial (mt) DNA H-strand promoter (HSP) produces a polycistronic precursor RNA encompassing all the genetic information encoded in that strand. Both mt rRNA genes and 12 out of 13 mRNA encoding genes are transcribed from HSP. There is a 20-fold higher expression of the rRNA gene region relative to the downstream mRNA encoding genes which involves an attenuation phenomenon just downstream of the 16S rRNA gene. A central role in this attenuation is played by mTERF, a protein that protects a 28 bp region immediately adjacent to the 3' end of the 16S rRNA gene. The mechanism and regulation of mTERF-dependent transcriptional termination is however still largely unknown.Two works have contributed interesting data, one by the applicant (Asin-Cayuela et al, JBC, 2004) and one by the host laboratory (Falkenberg et al, Nat Genet, 2002). The first one identifies an inactive trimeric form of mTERF, and also presents evidence for a role of mTERF on the regulation of transcription initiation. The second identifies two new mt transcription factors, TFB1M and TFB2M, that can support transcription when combined with the mt RNA polymerase and the mitochondrial transcription factor A. This finding will allow us to investigate the molecular function of mTERF in a defined in vitro system.Moreover the host laboratory has recently identified an mTERF paralogue in the human genome, TERF2. This protein is ubiquitously expressed, but little is known concerning its structure and function.The aims will be to : 1. Characterize the molecular function of mTERF in a pure in vitro system, in terms of termination activity, interaction with the transcription machinery both in transcription initiation and termination events, mechanism of monomer to trimer transition a nd comparison between HeLa and recombinant mTERF. 2. Investigate the dynamic interactions between mTERF and mtDNA,3. Characterize TERF2.

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