The long term objective is to study the mechanism of transcription of organelle DNA. The transcription system from chloroplasts presents an unique opportunity to define and analyze transcription of chloroplast (ct)DNA genes. A replication complex from chloroplasts has been obtained which contains ctDNA, RNA polymerase, DNA polymerase, DNA binding proteins, and 30-40 unidentified polypeptides. DNA bound complex synthesizes RNA in vitro which is transcribed from all the sequences of ctDNA. It is possible to remove endogenous ctDNA from this complex and use purified DNA as templates. We have isolated and cloned ribosomal RNA genes, tRNA genes, mRNA gene for the large subunit of ribulose-bis-phosphate carboxylase, and two photodependent genes from pea ctDNA. We now propose to analyze whether the RNA transcripts of the above genes are selectively and faithfully transcribed in vitro using complex containing endogenous ctDNA. These studies will be followed by analyzing the faithful transcription of these genes with the complex by removing the endogenous ctDNA and adding purified DNA gene templates. Finally, initiation and transcription of these genes will be studied with purified RNA polymerase and DNA gene templates. When we have analyzed the in vitro transcription of genes using the complex and the purified enzyme, it would be possible to design experiments to identify polypeptides which control the initiation, elongation and termination of ctRNA transcripts.
| Rajasekhar, V K; Sun, E; Meeker, R et al. (1991) Highly purified pea chloroplast RNA polymerase transcribes both rRNA and mRNA genes. Eur J Biochem 195:215-28 |
| Sun, E; Wu, B W; Tewari, K K (1989) In vitro analysis of the pea chloroplast 16S rRNA gene promoter. Mol Cell Biol 9:5650-9 |
