Identification of a putative mitochondrial RNA polymerase from Physarum polycephalum: characterization, expression, purification, and transcription in vitro.
Mitochondrial RNA polymerases (mtRNAPs) are necessary for the biogenesis of mitochondria and for proper mitochondrial function since they transcribe genes on mtDNA for tRNAs, rRNAs, and mRNAs. The unique type of RNA editing identified in mitochondria of Physarum polycephalum is thought to be closely associated with transcription, and as such, RNA editing activity would be expected to be closely associated with the mtRNAP. In order to better characterize the role of mtRNAPs in mitochondrial biogenesis and to determine the role of the Physarum mtRNAP in RNA editing, the cDNA of the Physarum mtRNAP was identified using PCR and degenerate primers designed from conserved motifs in mtRNAPs. This amplification product was used to screen a cDNA library for the cDNA corresponding to the Physarum mtRNAP. A cDNA corresponding to a 3.2 kb transcript containing a 997 codon open reading frame was identified. The amino acid sequence inferred from the open reading frame contains motifs characteristic of mtRNAPs. To confirm that a cDNA for an RNA polymerase had been isolated, the cDNA was expressed in E. coli as an N-terminal maltose binding protein (MBP) fusion protein. The fusion protein was purified by affinity chromatography and shown to have DNA-directed RNA polymerase activity. This functional mtRNAP will be useful for in vitro studies of mitochondrial transcription and RNA editing.
Miller
Mara L
ML
Department of Cell and Molecular Biology, The University of Texas at Dallas, 2601 N. Floyd Road, Richardson, TX 75080, USA.
Antes
Travis J
TJ
Qian
Fang
F
Miller
Dennis L
DL
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016839:Physarum polycephalum / Q000201:enzymology* / Q000235:genetics; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017393:RNA Editing / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic
D024101:Mitochondrial Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D012321:DNA-Directed RNA Polymerases