title: Expression and Purification of Mitochondrial RNA Polymerase and Transcription Factor A from Drosophila melanogaster.
abstract: Mitochondrial gene expression is essential in all organisms. Our understanding of mitochondrial transcription on a biochemical level has been limited by the inability to purify the individual protein components involved in mitochondrial gene expression. Recently, new systems have been identified that permit purification of these proteins from bacteria. However, the generalizability of these systems is not clear. Here, we have applied the technology from the Cameron lab to express and purify mitochondrial RNA polymerase and transcription factor A from Drosophila melanogaster. We show that the use of SUMO system to produce SUMO fusion proteins in bacteria is effective not only for the human and mouse proteins, but also for the fly proteins. The application of this system to produce the mitochondrial proteins from other organisms should permit detailed understanding of mitochondrial transcription from any organism.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 201 Althouse Laboratory, University Park, PA, 16802, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Gajewski
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 201 Althouse Laboratory, University Park, PA, 16802, USA.
authors.forename: Jamie J
authors.initials: JJ
authors.lastname: Arnold
authors.affiliation: Institute of Biosciences and Medical Technology, University of Tampere, Tampere, Finland.
authors.forename: Tiina S
authors.initials: TS
authors.lastname: Salminen
authors.affiliation: Institute of Biosciences and Medical Technology, University of Tampere, Tampere, Finland.|Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI, 48824, USA.
authors.forename: Laurie S
authors.initials: LS
authors.lastname: Kaguni
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 201 Althouse Laboratory, University Park, PA, 16802, USA. cec9@psu.edu.
authors.forename: Craig E
authors.initials: CE
authors.lastname: Cameron
mesh_terms: D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics*; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000235:genetics*; D004331:Drosophila melanogaster; D008928:Mitochondria / Q000235:genetics; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D000077278:RNA, Mitochondrial; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D029721:Drosophila Proteins; D000077278:RNA, Mitochondrial; D011993:Recombinant Fusion Proteins; C489029:TFAM protein, Drosophila; D014157:Transcription Factors; D012313:RNA; D012321:DNA-Directed RNA Polymerases; C000610357:mtRNApol protein, Drosophila; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease
keywords: Mitochondria; Mitochondrial RNA polymerase; Mitochondrial transcription; Protein expression; Protein purification; Transcription factor A