Identification and characterization of T3/T7 bacteriophage-like RNA polymerase sequences in wheat

Abstract

Using PCR-based methods, we assembled two wheat cDNA sequences, wheat-G and wheat-C, that encode T3/T7 bacteriophage-like RNA polymerases (RNAPs) sharing 45% amino acid identity. In phylogenetic analyses using maximum likelihood, parsimony and distance methods, the predicted protein sequence of wheat-G (1005 amino acids, 113 kDa) clusters with sequences of previously assigned mitochondrial RNAPs from dicotyledonous plants (Arabidopsis thaliana, Chenopodium album); likewise, in such analyses, the wheat-C sequence (949 amino acids, 107 kDa) affiliates specifically with the Arabidopsis sequence that encodes a phage-like RNAP thought to function in chloroplasts. To confirm biochemically the assignment of the gene encoding the putative wheat mitochondrial RNAP, we isolated a ca. 100 kDa wheat mitochondrial protein that is enriched in fractions displaying specific in vitro transcription activity and that reacts with an antibody raised against a recombinant maize phage-type RNAP. Internal peptide sequence information obtained from the 100-kDa polypeptide revealed that it corresponds to the predicted wheat-G cDNA sequence, providing direct evidence that the wheat-G gene (which we propose to call RpoTm) encodes the wheat mitochondrial RNAP.