Study Of Human Mitochondrial Transcription Factor B1 And B2 Function

The human mitochondrial transcription factor B1&2 are dual-functional proteins. They act as transcription factor in the mitochondrial genome trandcription. They can interact with the mitochondrial RNA polymerase (POLRMT) and the tiranscription factor A (TFA) which recgonize and bind to a specified sequence upstream the promotor, and form a trascription initiation complex. While, there was experiment suggested that, TFB1 was not necessarilly needed in the mitochondrial trascription initiation.There were a RNA methyltransferase motif on the C termimal domain of TFB1&2. We know very little about two proteins'RNA methyltransferase activity. We only know that TFB1 can bind with the methyl donor SAM, and TFB1 can act as a RNA methyltransferase in E.coli in which the homologous gene Ksg was knocked off.Schizosaccharomyces pombe is highly homologized with human. There are much more than 50 genes in S1. pombe homologous with human's disease related genes, which make S.pombe be a good research model for human disease. The dicovery of TFB1&2 were related to the homologous protein MTF1 in S. pombe.To find out the functional relationship between TFB1&2 and MTF1, and to research the RNA methyltransferase activity of the two protein, we do experiments as follows:1. The fuctional similarity between TFB1&2 and MTF1:the pREP3-tfb1&2-GFP fusion plasmids were constucted and transformed in to fission yeast (Schizosacch-aromyces pombe), we found the TFB1&2 were localized in mitochondrial through flouresence microscopy; we got TFB1&2 overexpression strains through construction of pREP3-tfb1&2 and transformation them in to yeast, then we detected the effect of the TFB1&2 overexpression on the fission yeast's mitochondrial gene; the TFB1 &2 were putified in E.coli, and we found that both of TFB1&2 can bind with the fission yeast's mitchonchondrial RNA polymerase Rpo41 through pull-down experiment.2. The research of RNA methyltransferase activity:we found that both of TFB1&2 can bind with h-mt12S rRNA which is the enzyme's substrace RNA; we found that TFB1 can form homologous polymer through pull-down experiment.