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.

Mitochondrial gene expression is necessary for proper mitochondrial biogenesis. Genes on the mitochondrial DNA are transcribed by a dedicated mitochondrial RNA polymerase (mtRNAP) that is encoded in the nucleus and imported into mitochondria. In the myxomycete Physarum polycephalum, nucleotides that are not specified by the mitochondrial DNA templates are inserted into some RNAs, a process called RNA editing. This is an essential step in the expression of these RNAs, as the insertion of the nontemplated nucleotides creates open reading frames for the production of proteins from mRNAs or produces required secondary structure in rRNAs and tRNAs. The nontemplated nucleotide is added to the 3 end of the RNA as the RNA is being synthesized during mitochondrial transcription. Because RNA editing is cotranscriptional, the mtRNAP is implicated in RNA editing as well as transcription. We have cloned the cDNA for the mtRNAP of Physarum and have expressed the mtRNAP in Escherichia coli. We have used in vitro transcription assays based on the Physarum mtRNAP to identify a novel activity associated with the mtRNAP in which non-DNA-templated nucleotides are added to the 3 end of RNAs. Any of the four ribonucleoside triphosphates (rNTPs) can act as precursors for this process, and this novel activity is observed when only one rNTP is supplied, a condition under which transcription does not occur. The implications of this activity for the mechanism of RNA editing are discussed.RNA editing is an additional step in gene expression that involves the changing of the RNA sequence relative to the DNA sequence that encodes it (3, 4). RNA editing is a required step in the expression of genes in the mitochondria of Physarum polycephalum. It involves the insertion of nontemplated nucleotides into the RNA (28). In Physarum mitochondrial RNA editing, the nucleotide that is inserted can be any of the four ribonucleotides, and the insertion of ribonucleotides is seen in all three types of RNA, mRNA, tRNA, and rRNA. In mRNAs, the insertion of nucleotides will invariably create an open reading frame from which a protein can be expressed (16,28), and in tRNAs and rRNAs, it will create the necessary structures required for the proper function of these molecules (1,27,32).Insertional RNA editing can fall into two distinct types based on the mechanism employed: posttranscriptional and cotranscriptional. Posttranscriptional RNA editing involves (i) the release of the RNA from the RNA polymerase (RNAP), (ii) the cleavage of the RNA backbone with an RNA endonuclease, (iii) the addition of a nontemplated nucleotide with a terminal transferase-like activity, and (iv) the ligation of the RNA with an RNA ligase to restore the RNA backbone. This posttranscriptional type of RNA editing is seen in trypanosome mitochondrial mRNAs, in which uridine nucleotides are inserted in or deleted from the RNA relative to the DNA template, creating open reading frames from which proteins can be produced (15,17).In contrast, cotranscriptional RNA editing involves the add...