The nucleotide sequences and expression of genes for the beta and epsilon subunits of ATP synthase from rice (Oryza sativa L.)

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1 Jpn. J. Genet. (1989) 64, pp The nucleotide sequences and expression of genes for the beta and epsilon subunits of ATP synthase from rice (Oryza sativa L.) Yoko NIsxtzAwA1 and Atsushi HIRAI* Graduate Division of Biochemical Regulation, Faculty of Agriculture, Nagoya University, Nagoya J64-O1 (Received,2,2 February 1989) ABSTRACT The genes for the j3 and e subunits of ATP synthase were cloned from rice chloroplast DNA. The nucleotide sequences and the deduced amino acid sequences of these genes were determined. The site of initiation of transcription was examined by S1-mapping analysis. The relation between stability of mrna and that of a stem and loop structure at the 3' end of mrna is discussed. 1. INTRODUCTION ATP synthase is an important participant in photosynthesis, and five of its eight subunits are encoded by chloroplast DNA in higher plants (Shinozaki et al., 1986). The genes for the j9 and e subunits (atpb and atpe, respectively) of ATP synthase are located close to the gene for the large subunit of RuBisCO, another important participant in photosynthesis, but are transcribed in the opposite directions (Krebbers et al., 1982). Although both ATP synthase and RuBisCO are key enzymes in photosynthesis, they are distinct in terms of the following characteristics. ATP synthase is expressed in both mesophyll and bundle sheath cells in C4 plants, such as maize, while RuBisCO is not expressed in mesophyll cells in C4 plants (Link et al., 1978). However, when RuBisCO is expressed in any plant cell, the level of expression of RuBisCO is much greater than that of ATP synthase. Furthermore, it has been reported that the efficiency of atpb transcription is affected by the rbcl promoter region because of close spacing of the two genes (Hanley-Bowdoin and Chua, 1989). Thus, in order to understand the mechanism of chloroplast gene expression, it is of interest to accumulate the data for the fine structure of two genes and the intercistronic region between them and the expression of two genes from several plants. We have recently reported the nucleotide sequence and S1 mapping data for the large subunit of RuBisCO from rice, and we found a possible C4-specific deletion 9 1 Present address: Natinoal Institute of Agrobiological Resources *Corresponding author., Tsukuba 305, Japan.

2 224 Y. NISHIZAWA and A. HIRAI bases upstream from the "--35" region, using our data and those of others (Nishizawa and Hirai, 1987). Therefore, it is also of interest to compare the sequences of the promoter regions of the subunit of ATP synthase from maize and rice, a protein whose expression is same in C3 and C4 plants. In this paper, we report nucleotide sequences of the atpb/e genes from rice, and demonstrate that the nucleotide sequences in the promoter regions of the genes from rice and maize are identical. We also show that a stem and loop structure at the 3' end of the mrna is less stable than the corresponding structures in the stable mrnas transcribed from the rbcl and psba genes in chloroplasts. 2. MATERIALS AND METHODS Pst-14 and Hind-1 fragments of chloroplast DNA from rice (Hirai et al., 1985) were cloned in puc8 and puc18 respectively. These plasmid DNAs were digested with restriction enzyme(s) and subcloned into M13mp10 or M13mp11. The nucleotide sequence was determined by the dideoxy method (Sanger et al., 1977). S1 mapping was carried out by the method of Maniatis et al. (1983), using rice total leaf RNA. The free energy of stem and loop structures was calculated by the method of Tinoco et al. (1973). Nucleotide sequence of atpb and atpe 3. RESULTS AND DISCUSSION The nucleotide sequence and the deduced amino acid sequence of atpb and atpe are shown in Fig. 1. The j9 and e subunits of ATP synthase from rice contain 498 and 137 amino acids, respectively. Furthermore the two genes overlap by 4 bp, as they do in other plants (Krebbers et al., 1982). As shown in Table 1, homologies in nucleotide and amino acid sequences of the two subunits among four important cereal plants are similar. Therefore, it is interesting that the amino acid sequence of the ~9 subunit from rice shows 91.6% homology to the sequence of the j3 subunit from tobacco, while the e subunit shows only 72.5% homology to the amino acid sequence of the subunit from tobacco (Shinozaki et al., 1983). Promoter site of atpb We performed an analysis by S1 mapping to determine the site of initiation of transcription and to locate the promoter region of the atpb gene. As illustrated in Fig. 2, our results show that transcription begins about 340 bases upstream from a SmaI site, i.e. about 310 bases before the atpb protein-coding region.

3 Nucleotide sequence of atpb/e in rice chloroplasts 225 Fig. 1. Nucleotide sequence of the atpb and atpe overlapping genes from rice and the deduced amino acid sequences. Possible "-35" and "-10" sequences are boxed, and the tentative starting position for transcription is shown by an arrow. Putative ribosome-binding sites are indicated by double underlining, and the sequence at the 3" end, which can form a stem and loop structure, is shown by a pair of single lines under the nucleotide sequence.

4 226 Y. NISHIZAWA and A. HIRAI Table 1. Sequence homologies among atpb and atpe from cereal crops 1, Howe et al. (1985); Shinozaki et al. (1983). 2, Zurawski and Clegg (1984) 3, Krebbers et al. (1982) 4,

5 Nucleotide sequence of atpb/e in rice chloroplasts 227 Fig. 3. Comparison of the 5' and tobacco. flanking sequences of atpb from four cereal plants The "-35" and "-10" sequences are found in the upstream region, as shown in Fig. 1. The promoter sequences of atpb from rice and other monocot crop plants are compared in Fig. 3, which shows that the sequences from the various plants are almost identical. Furthermore the maize-specific 5 base deletion, which was found in the upstream of the promoter region of the large subunit gene for Fig. 4. Possible stem and loop strucutres at the 3' end of the atpb/e, psba genes. Postitions are given relative to the respective TGA colon. rbcl and Fig. 2. Determination of the 5` terminus of the mrna transcribed from the atpb/e gene by S1-mapping analysis. a: Radioautograph of S1-protected fragments. Incubation times and temperatures are shown at the top of the gels. The sizes of the radioactive marker fragments are indicated (in kb) at the right of the gels. b: The fragment used as a probe.

6 228 Y. NISHIZAWA and A. HIRAI RuBisCO (Nishizawa and Hirai, 1987), has not been detected in the corresponding position of the atpb gene in maize. Structure of the 3' end of atpb and atpe A transcriptional terminator-type stem and loop structure was found about 50 bases downstream from the stop colon. The free energy of this structure, when transcribed into RNA, was calculated to be kcal/mol. This value shows that the structure is less stable than the free energies of analogous structures in =rbcl or =psba, as shown in Fig. 4. Stern and Gruissem (1987) proposed that a stem and loop structure acts to protect mrna from degradation. Therefore, this unstable structure may reflect the potential instability of the mrna for the ~9 and e subunits, and may explain the low level of synthesis of these proteins. We thank Drs. M. Sugiura and J. Hiratsuka, Center for Gene Research, Nagoya University for technical advice and encouragement. This work was partly supported by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan. REFERENCES HANLEY-BOWDOI, N, L. and CHUA, N. H. (1989) Transcriptional interaction between the promoters of the maize chloroplast genes which encode the subunit of ATP synthase and the large subunit of ribulose 1,5-bisphosphate carboxylase. Mol. Gen. Genet. 215, HIRAI, A., ISHIBASHI, T., MORIKAMI, A., IWATSUKI, N., SHINOZAKI, K. and SUGIURA, M. (1895) Rice chloroplast DNA: a physical map and the location of the genes for the 32KD photosystem II reaction center protein. Theor. Appl. Genet. 70, HOWE, C. J., FEARNLEY, I. M., WALKER, J. E., DYER, T. A, and GRAY, J. C. (1985) Nucleotide sequences of the genes for the alpha, beta and epsilon subunits of wheat chloroplast ATP synthase. Plant Mol. Biol. 4, KANNO, A. and HIRAI, A. (1989) The nucleotide sequence and expression of the gene for the 32 kd quinone-binding protein form rice. Plant Science 59, KREBBERS, E. T., LARRINUA, I. M., MCINTOSH, L. and BOGORAD, L. (1982) The maize chloroplast genes for the 9 and E subunits of the photosynthetic coupling factor CF1 are fused. Nucleic Acids Res. 10, LINK, G., COEN, D. M., and BOGORAD, L. (1987) Differential expression of the gene for the large subunit of ribulose bisphosphate carboxylase in maize leaf cell types. Cell 15, MANIATIS, T., FRITSCH, E. F, and SAMBROOK, J. (1983) Molecular Cloning. A Laboratory Manual, p Cold Spring Harbor Lab. Cold Spring Harbor, New York. NISHIZAWA, Y. and HIRAI, A. (1987) Nucleotide sequence and expression of the gene for the large subunit of rice ribulose 1, 5-bisphosphate carboxylase. Jpn. J. Genet. 62, SANGER, F., NICKLEN, S. and COULSON, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, SHINOZAKI, K., DENO, H., KATO, A. and SUGIURA, M. (1983) Overlap and cotranscription of the genes for the beta and epsilon subunits of tobacco chloroplast ATPase. Gene 24, SHINOZAKI, K., OHMS, M., TANAKA, M., WAKASUGI, T., HAYASHIDA, N., MATSUBAYASHI, T., ZAITA, N., CHUNWONGSE, J., OBOKATA, J., YAMAGUCHI-SHINOZAKI, K., OHTO, C., TORAZAWA, K., MENG, B. Y., SUGITA, M., RENO, H., KAMOGASHIRA, T., YAMADA, K., KUSUDA, J., TAKAIWA, F., KATO, A., ToHDOH, N., SHIMADA, H., and SUGIURA, M.; (1986) The complete nucleotide sequence of the

7 Nucleotide sequence of atpb/e in rice chloroplasts 229 tobacco cholroplast genome: its gene organization and expression. EMBO J. 5, STERN, D. B. and GRUISSEM, W. (1987) Control of plastid gene expression: 3' inverted repeats act as mrna processing and stabilizing elements, but do not terminate transcription. Cell 51, TINOCO, I., BORER, P. N., DENGLER, B. and LEVINE, M. D. (1973) Improved estimation of secondary structure in ribonucleic acids. Nature New Biol. 246, ZURAWSKI, G. and CLEGG, M. T. (1984) The barley chloroplast DNA atpbe, trnm2, and trnvl loci. Nucleic Acids Res. 12,