Analyses of the Extent of Immunological Relatedness between a Highly Purified Pea Chloroplast Functional RNA Polymerase and Escherichia coli RNA Polymerase

The reported nucleotide sequence homology between the RNA polymerase (EC 2.7.7.6) coding (rpo) genes in Escherichia coli and their putative homologues in chloroplast DNA is not only limited to about 26-50 %, but also scattered. Utilising the homologous in vitro transcription system and immunological approach, we address as to which extent the chloroplast functional RNA polymerase and E. coli RNA polymerase are related. Highly purified RNA polymerase from pea chloroplasts has been demonstrated to transcribe both ribosomal and messenger RNA genes and the purified homologous antibodies inhibit the chloroplast gene promoter recognizing ability as well as in vitro transcriptional activity of the enzyme [Rajasekhar et al., 1991]. While recognizing all of the polypeptides of the chloroplast enzyme on Western blots, the chloroplast RNA polymerase antibodies did not cross-react with the sub-units of E. coli RNA polymerase or wheat germ RNA polymerase and vice versa. Antibodies against the 130 kDa,110 kDa, 75-95 kDa and 48 kDa polypeptides of the purified chloroplast RNA polymerase immobilized only homologous RNA polymerase on the affinity columns. The active enzyme eluted from the immunoaffinity columns showed sub-unit patterns similar to that of purified RNA polymerase. The specificity of these antibodies was confirmed with the lack of immobilization of E. coli RNA polymerase and vice versa with the E. coli RNA polymerase antibodies. The E. coli RNA polymerase also manifested transcription in vitro utilising the supercoiled 7A/30T recombinant containing the chloroplast 16S rRNA minigene as well as the supercoiled pCB 1-3 recombinant containing the upstream sequences of ribulose-1,5-bisphosphate carboxylase large sub-unit (rbc-L) gene. The transcriptional activities, however, were specifically affected only by homologous RNA polymerase antibodies, but not by heterologous RNA polymerase antibodies. While both the chloroplast and the E. coli RNA polymerases caused mobility shifts of the chloroplast 16S rRNA gene and rbc-L gene promoter fragments, only the homologous antibodies were effective in abolishing this response. The data obtained form the first functional evidence that the highly purified pea chloroplast RNA polymerase and the E. coli RNA polymerase are immunologically only distantly related.