| Although mitochondrial promoters have been well defined in vascular plants, relatively little is known of the transcriptional machinery. To characterize the transcription initiation complex at the maize mitochondrial atpA promoter, exonuclease III was used to map the borders of DNA-protein interactions. These experiments revealed that with a wild-type promoter, protein factors occupy as much as 36 bp, from positions --20 to +16 relative to the transcription initiation site.;Biochemical studies using an antibody raised against the C-terminus of RpoTp identified mitochondrial polypeptides of approximately 100 kD in a transcriptionally active fraction. Their presence was correlated with RNA polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. Immunoblot analysis and an antibody-inked polymerase assay, together with the in vivo localization, indicated that rpoTp specifies a plastid RNA polymerase component.;Although it is not clear that how many protein factors are required for specific transcription initiation in higher plant mitochondria, several observations suggested that the machinery is similar to that of yeast, which consists of two proteins. One of these proteins is a core RNA polymerase of the T7 family, while the other factor has limited sequence homology, and strong functional homology, to bacterial sigma factors. Two possible plant mitochondrial transcription factors, ZmSig2 and p63, were proposed based on their resemblance to bacterial sigma factors, and localization. In an attempt to reconstitute transcription initiation, we have overexpressed ZmSig2 and p63 in E. coli, and purified the proteins.;As a first step to characterize the maize mitochondrial transcription machinery, we obtained two maize cDNA clones, rpoTm and rpoTp, that encode putative T7-like RNA polymerases. The predicted proteins, RpoTm and RpoTp, share 54% amino acid sequence identity, are nucleus-encoded, and are probably encoded by single copy genes. Sequence alignments, among several T7-like RNA polymerases revealed at least 11 conserved domains as well as conservation of some functional motifs and important catalytic residues; suggesting that rpoTm and rpoTp encoded functional RNA polymerase. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that the rpoTm and rpoTp encoded proteins are targeted exclusively to mitochondria and plastids, respectively. |
