Identification of a putative mitochondrial RNA polymerase from Physarum polycephalum: characterization, expression, purification, and transcription in vitro

Abstract

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.

Organization and expression of organellar genomes

“…Sequences encoding such an enzyme have been specifically amplified from many protists (Cermakian et al 1996;Li et al 2001) and can typically be found in sequenced protist nuclear genomes. The corresponding activity has been experimentally verified in some lineages (Grams et al 2002;Miller et al 2006). However, as noted above, the mitochondrial genomes of R. americana and other jakobids encode a bacterial-type RNA polymerase (Gray et al 2004).…”

Distinct roles for sequences upstream of and downstream from Physarum editing sites

“…Overall core structures are well conserved, even between single and multi-subunit polymerases. Although there are currently no equivalent structures for the mitochondrial RNA polymerase from Physarum polycephalum (Pp mtRNAP), based on its high degree of homology with the bacteriophage T7 RNA polymerase (T7 RNAP) (Miller et al 2006;Gott and Rhee 2007), the basic features of the Pp mtRNAP elongation complex are likely to be similar. Of particular interest to our findings here is that in the T7 RNAP elongation complex, sequences immediately upstream of the point of nucleotide addition are involved in the 7-8-bp RNA:DNA hybrid and the z9-nt transcriptional bubble, while roughly 8 bp of downstream DNA lies against a positively charged patch on the exterior of the polymerase (Tahirov et al 2002;Steitz 2002, 2004;Temiakov et al 2004).…”

RNA editing of 10 Didymium iridis mitochondrial genes and comparison with the homologous genes in Physarum polycephalum

“…Recently, the P. polycephalum mitochondrial RNA polymerase has been cloned (Miller et al 2006), and it has been found to add non-DNA-templated nucleotides to the 39 end of RNAs (Miller and Miller 2008). Using chimeric templates and in vitro mitochondrial transcription elongation complexes Rhee et al (2009) demonstrated the importance of 9 base pairs (bp) to either side of C insertion sites in directing this type of editing event.…”