Expression and purification of wild type and mutant forms of the yeast mitochondrial core RNA polymerase, Rpo41.
The mitochondrial RNA polymerase (mtRNAP) from Saccharomyces cerevisiae (yeast) is composed of two nuclear encoded proteins, the core RNA polymerase (Rpo41) and the mitochondrial transcription factor (Mtf1). Although Rpo41 is strikingly similar to the single subunit RNAPs from the T7 and T3 bacteriophage (T7RNAP), the core mtRNAP requires Mtf1 for accurate transcription from a linear promoter-containing DNA template, while T7RNAP does not require any other additional factors for promoter selectivity. The fact that the mtRNAP requires an additional promoter utilization factor makes it an excellent model system for the analysis of the transitions that occur during transcription initiation. However, large-scale purification of the 153 kDa Rpo41 has only been reported from yeast cells, or as a recombinant from baculovirus, both sources requiring extensive purification with poor yields. We have developed a His-tagged Rpo41 expression construct suitable for rapid purification of large amounts of soluble Rpo41 from bacterial cells. Transcriptionally active forms of both wild type and point mutants of Rpo41 can be purified by a combination of batch ion exchange chromatography to remove nucleic acids and nickel affinity chromatography. An additional advantage of the isolation of Rpo41 from bacterial cells is the absence of its associated specificity factor Mtf1. This allows analysis of combinations of mutant forms of both components of the mtRNAP holoenzyme.
Matsunaga
Michio
M
Department of Biochemistry and Molecular Genetics, and Molecular Biology Program, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Jang
Sei-Heon
SH
Jaehning
Judith A
JA
D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; C487996:RPO41 protein, S cerevisiae