| Mitochondrial RNA polymerase (mtRNAP) is responsible for transcription of mitochondrial DNA. In Physarum polycephalum mitochondrial RNA, high level of RNA editing was observed during transcription. The mitochondrial RNA polymerase is involved in this process, but little is known regarding its mechanism. To understand mechanisms of transcription and RNA editing activity of Physarum polycephalum mitochondrial RNA polymerase, we have initiated structural studies of this enzyme using the method of X-ray crystallography. This process requires access to large quantity of the pure protein for crystallization trials. This thesis reports generation of expression construct, protein purification procedures, and crystallization of the Physarum polycephalum mitochondrial RNA polymerase. The protein was produced as a ubiquitin fusion protein to increase its solubility then purified using a salting out procedure, ion exchange and size exclusion chromatography. Small needle-like crystals were obtained. The identity of the protein was confirmed through Mass Spectrometry analyses. |
