| Organization and expression of chloroplast genomes
Kazuo SHINOZAKI and Masahiro SUGIURA National Institute of Genetics, Mishima 411, Japan Recent progress in molecular biological analyses of chloroplast DNAs have revealed many interesting features of these genomes. Chloroplast DNAs of higher plants are double-stranded circular DNA with about 160,000 base-pairs (bp) Iength, and have a long inverted-repeat sequence (22,000 bp) (Fig. 1). Approximately 100-200 genes are thought to be encoded in chloroplast DNA. In this article, we refer to organization and expression of genes for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase, rRNA and tRNA of tobacco chloroplast. We have cloned and sequenced the LS gene to tobacco chloroplast and its flanking regions. The coding region contains 1431 nucleotides (477 codons). Sequences of the LS coding regions are highly conserved among higher plants and resemble quite well each other (about 90% homology). The positions in the gene corresponding to the 5' and the 3' ends of tobacco LS mRNA have been located by the S1 nuclease mapping procedure. The promoter sequence of the LS gene has homology with Escherichia coli promoter sequences; its terminator sequence is capable of forming a stem-and-loop structure. A sequence GGAGG, which is complementary to a sequence near the 3' end of tobacco chloroplast 16S rRNA and a putative ribosome binding site, occurs 6-10 bp upstream from the initiation codon. These observations suggest that molecular mechanisms of transcription and translation of chloroplast are similar to those of prokaryotes. In fast, we demonstrated the E. coli RNA polymerase can start the LS mRNA in vitro at the same position as that of in vivo. We have also determined the nucleotide sequence of rRNA operon of tobacco chloroplast. Two sets of rRNA operon are located on inverted-repeat sequences (Fig. 1). The order of genes of rRNA operon is tRNAval-16S rRNA-tRNAIle-tRNAAla-23S rRNA-4.5S rRNA-5S rRNA. The coding regions of 16S, 23S, 4.5S and 5S rRNA genes are 1485, 2804, 103 and 121 base pairs long. Nucleotide sequences of 16S and 23S rRNA have approximately 70% homology with those of E. coli. The 3' terminal region of 16S rRNA gene contains the sequence CCTCC which is complementary to sequence found at the 5' terminus of the LS mRNA. The 4.5S rRNA is found in the large subunit of the chloroplast ribosomes, and its nucleotide sequence has homology with that of 3' end of 23S rRNA. The nucleotide sequence of 5S RNA from tobacco chloroplast resemblaes well that from blue-green algae, which support the idea of the symbiotic origin of chloroplast genomes. The region preceding the 16S rRNA gene contains tRNAVal (GAC) gene and promoter-type sequences similar to those which occur in E. coli. E. coli RNA polymerase can recognize these sequences and co-transcribes the tRNAVal and rRNA genes in vitro. The spacer region between the 16S and 23S rRNA genes is 2080 bp long and contain two tRNA genes; tRNAIle and tRNAAla. These two tRNA genes have large introns of about 700 bp in their anticodon loops. They are co-transcribed with 16S and 23S rRNA genes as a single precursor RNA of 8.2 kb. A Iong intron is also found in tRNAVal (UCA) gene which is located near the LS gene. The CCA sequence, which is coded for E. coli tRNA genes, is not found at 3' ends of tRNA genes of chloroplast. These obervations reveal eukaryotic features of tRNA genes of chloroplasts. |