Expression, Purification And Crystallization Of The Mitochondrial Core RNA Polymerase, Rpo41 And Transcription Factor, Mtf1 In Saccharomyces Pombe

Mitochondria are the energy-transducing organelles of eukaryotic cells in which fuels to drive cellular metabolism are converted into ATP through the process of oxidative phosphorylation. Mitochondria contain a separate genome (mtDNA) that encodes several mRNAs and the rRNAs and tRNAs required for their translation into components of the oxidative phosphorylation system. These mitochondrial transcripts are synthesized by nuclear DNA-encoded RNA polymerase (RNAP) subunits distinct from those found in the nucleus of the eukaryotic cell. The yeast(Saccharomyces pombe) transcription machinery consists of the core RNAP subunit, a 127 kDa protein called Rpo41, and the transcription factor, a 42 kDa protein called Mtfl. The Rpo41 is evolutionarily related to the single-subunit phage T7/T3 RNAP. And the crystal structure determination of Mtfl has revealed a significant structural similarity with the family of RNA methyltransferases. Furthermore, Mtfl and its functional homologues from multicellular organisms all share amino acid sequence similarity with RNA methyltransferases. Both proteins are transported into the mitochondria where they form a heterodimeric Rpo41/Mtfl complex (mtRNAP).In order to know the manner in which the Rpo41-Mtfl complex recognizes the promoter and initiates transcription, we expressed, purified the complex. We also crystallized this complex with the mitochondrial promoter. Finally, we got the crystal. The optimization of this crystal is ongoing.