The long term objective is to study the mechanism of transcription of chloroplast DNA genes using homologous RNA polymerase. We have purified an RNA polymerase preparation from pea chloroplasts that shows strong preference to chloroplast DNA templates and can synthesize in vitro full length rRNA, tRNA, and photogene 32 mRNA transcripts using cloned genes. We now propose to study the following: 1) A detailed study on the nucleotide sequences required (promoter sequences) for recognition by the RNA polymerase for all three types of RNA chains i.e. rRNA, tRNA, and mRNA. These experiments will be carried out by constructing deletion mutants of the sequences that surround transcription initiation sites of genes using Ba131. Identification of the sequences at the 5' end of genes required for the transcription will be followed by experiments where single and multiple point mutations within the control region will be produced and the mutated DNA studied for transcription. These experiments will enable us to precisely identify the nucleotides that constitute specific promoters of each type of gene; 2) An analysis of nucleotide sequences at the 3' end of the genes that might be involved in the temination of transcription; 3) Further purification of the RNA polymerase enzyme using conventional purification procedures to establish whether there is a single RNA polymerase that transcribes different types of chloroplast DNA genes or there are multiple polymerases each specific for ribosomal RNA, transfer RNA, and messenger RNA genes; and 4) Analyze the structure of RNA polymerase(s) using monoclonal antibodies. These studies will, in future, be followed by experiments that would identify polypeptides which control the initiation, elongation and termination of chloroplast RNA 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 |
