Summary
The gene rpoA, encoding a protein homologous to the alpha subunit of RNA polymerase from Escherichia coli has been located in pea chloroplast DNA downstream of the petD gene for subunit IV of the cytochrome b-f complex. Nucleotide sequence analysis has revealed that rpoA encodes a polypeptide of 334 amino acid residues with a molecular weight of 38916. Northern blot analysis has shown that rpoA is co-transcribed with the gene for ribosomal protein S11. A lacZ-rpoA gene-fusion has been constructed and expressed in E. coli. Antibodies raised against the fusion protein have been employed to demonstrate the synthesis of the rpoA gene product in isolated pea chloroplasts. Western blot analysis using these antibodies and antibodies against the RNA polymerase core enzyme from the cyanobacterium, Anabaena 7120, has revealed the presence of the gene product in a crude RNA polymerase preparation from pea chloroplasts.
Similar content being viewed by others
References
Bedwell D, Davis G, Gosink M, Post L, Nomura M, Kestler H, Zengel JM, Lindahl L (1985) Nucleotide sequence of the alpha ribosomal protein operon of Escherichia coli. Nucleic Acids Res 13:3891–3903
Biggin M, Farrell PJ, Barrell BG (1984) Transcription and DNA sequence of the BamHI L fragment of B95-8 Epstein-Barr virus. EMBO J 3:1083–1090
Biggin MD, Gibson TJ, Hong GF (1983) Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci USA 80:3963–3965
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Blair GE, Ellis RJ (1973) Protein synthesis in chloroplasts. I. Light-driven synthesis of the large subunit of Fraction I protein by isolated pea chloroplasts. Biochim Biophys Acta 319:223–234
Bonner WM, Laskey RA (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46:83–88
Briat J-F, Bisanz-Sever C, Laulhère J-P, Lerbs S, Lescure A-M, Mache R (1987) The RNA polymerase from chloroplasts and its use for in vitro transcription of plastid genes. Plant Physiol Biochem 25:273–281
Bülow S, Link G (1988) Sigma-like activity from mustard (Sinapis alba L.) chloroplasts conferring DNA-binding and transcription specificity to E. coli RNA polymerase. Plant Mol Biol 10:349–357
Bünger W, Feierabend J (1980) Capacity for RNA synthesis in 70 S ribosome-deficient plastids of heat-bleached rye leaves. Planta 149:163–169
Burgess RR (1976) Purification and physical properties of E. coli RNA polymerase. In: Losick R, Chamberlin M (eds) RNA polymerase. Cold Spring Harbor Laboratories. Cold Spring Harbor, New York, pp 69–100
Cohen SN, Chang ACY, Hsu L (1972) Non-chromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114
Covey SN, Hull R (1981) Transcription of cauliflower mosaic virus DNA. Detection of transcripts, properties and location of the gene encoding the virus inclusion body protein. Virology 111:463–474
Cushman JC, Hallick RB, Price CA (1988) The two genes for the P700 chlorophyll a apoprotein on the Euglena gracilis chloroplast genome contain multiple introns. Curr Genet 13:159–171
Ellis RJ, Hartley MR (1971) Sites of synthesis of chloroplast proteins. Nature New Biol 233:193–196
Greenberg BM, Narita JO, De Luca-Flaherty C, Gruissem W, Rushlow KA, Hallick RB (1984) Evidence for two RNA polymerase activities in Euglena gracilis chloroplasts. J Biol Chem 259:14880–14887
Greenberg BM, Narita JO, De Luca-Flaherty C, Hallick RB (1985) Properties of chloroplast RNA polymerases. In: Steinback KE, Bonitz S, Arntzen CJ, Bogorad L (eds) Molecular biology of the photosynthetic apparatus. Cold Harbor Spring Laboratory, Cold Spring Harbor, New York, pp 303–310
Howe CJ, Bowman CM, Dyer TA, Gray JC (1982) Localisation of wheat chloroplast genes for the beta and epsilon subunits of ATP synthase. Mol Gen Genet 186:525–530
Hudson GS, Holton TA, Whitfeld PR, Bottomley W (1988) Spinach chloroplast rpoBC genes encode three subunits of the chloroplast RNA polymerase. J Mol Biol 200:639–654
Kieny MP, Lathe R, Lecocq JR (1983) New versatile cloning and sequencing vectors based on bacteriophage M13. Gene 26:91–99
Kohchi T, Yoshida T, Komano T, Ohyama K (1988) Divergent mRNA transcription in the chloroplast psbB operon. EMBO J 7:885–891
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685
Lam E, Chua N-H (1987) Chloroplast DNA gyrase and in vitro regulation of transcription by template topology and novobiocin. Plant Mol Biol 8:415–424
Lerbs S, Bräutigam E, Mache R (1988) DNA-dependent RNA polymerase of spinach chloroplasts: characterization of α-like and σ-polypeptides. Mol Gen Genet 211:459–464
Lerbs S, Bräutigam E, Parthier B (1985) Polypeptides of DNA-dependent RNA polymerase of spinach chloroplasts: characterization by antibody-linked polymerase assay and determination of sites of synthesis. EMBO J 4:1661–1666
McMaster GK, Carmichael GG (1977) Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci USA 74:4835–4838
Newman BJ, Gray JC (1988) Characterisation of a full-length cDNA clone for pea ferredoxin-NADP+ reductase. Plant Mol Biol 10:511–520
Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi H, Ozeki H (1986) Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322:572–574
Orozco EM, Mullet JE, Chua N-H (1985) An in vitro system for accurate transcription initiation of chloroplast protein genes. Nucleic Acids Res 13:1283–1302
Ovchinnikov YA, Lipkin VN, Modyanov NN, Chertov OY, Smirnov YV (1977) Primary structure of α-subunit of DNA-dependent RNA polymerase from Escherichia coli. FEBS Lett 76:108–111
Palmer JD, Thompson WF (1981) Rearrangements in the chloroplast genomes of mung bean and pea. Proc Natl Acad Sci USA 78:5533–5537
Palmer JD, Osorio B, Thompson WF (1988) Evolutionary significance of inversions in legume chloroplast DNAs. Curr Genet 14:65–74
Phillips AL, Gray JC (1984) Location and nucleotide sequence of the gene for the 15.2 kDa polypeptide of the cytochrome b-f complex from pea chloroplast. Mol Gen Genet 194:477–484
Purton S, Gray JC (1987a) Nucleotide sequence of the gene for ribosomal protein S11 in pea chloroplast DNA. Nucleic Acids Res 15:1873
Purton S, Gray JC (1987b) Nucleotide sequence of the gene for ribosomal protein L36 in pea chloroplast DNA. Nucleic Acids Res 15:9080
Ruf M, Kössel H (1988) Structure and expression of the gene coding for the α-subunit of DNA-dependent RNA polymerase from the chloroplast genome of Zea mays. Nucleic Acids Res 16:5741–5754
Sanger F, Coulsen AR, Barrell BG, Smith AJH, Roe BA (1980) Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143:161–178
Schneider GJ, Tumer NE, Richaud C, Borbely G, Haselkorn R (1987) Purification and characterization of RNA polymerase from the cyanobacterium Anabaena 7120. J Biol Chem 262:14633–14639
Shinozaki J, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, Deno H, Kamagashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M (1986) The complete sequence of the tobacco chloroplast genome: its organization and expression. EMBO J 5:2043–2049
Siemenroth A, Wollgiehn R, Neumann D, Börner Th (1981) Synthesis of ribosomal RNA in ribosome-deficient plastids of the mutant “albostrians” of Hordeum vulgare L. Planta 153:547–555
Sijben-Müller G, Hallick RB, Alt J, Westhoff P, Herrmann RG (1986) Spinach plastid genes coding for initiation factor IF-1, ribosomal protein S11 and RNA polymerase α-subunit. Nucleic Acids Res 2:1029–1044
Stanley KK, Luzio JP (1984) Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO J 3:1429–1434
Stern DB, Gruissem W (1987) Control of plastid gene expression: 3′ inverted repeats act as mRNA processing and stabilizing elements but do not terminate transcription. Cell 51:1145–1157
Suh J-W, Boylan SA, Price CW (1986) Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal gene cluster. J Bacteriol 168:65–71
Surzycki SJ, Shellenbarger DL (1976) Purification and characterization of a putative sigma factor from Chlamydomonas reinhardii. Proc Natl Acad Sci USA 73:3961–3965
Woodbury NW, Roberts LL, Palmer JD, Thompson WF (1988) A transcription map of the pea chloroplast genome. Curr Genet 14:75–89
Yen TSB, Webster RE (1981) Bacteriophage f1 gene II and X proteins. J Biol Chem 256:11259–11265
Author information
Authors and Affiliations
Additional information
Communicated by D. M. Lonsdale
Rights and permissions
About this article
Cite this article
Purton, S., Gray, J.C. The plastid rpoA gene encoding a protein homologous to the bacterial RNA polymerase alpha subunit is expressed in pea chloroplasts. Mol Gen Genet 217, 77–84 (1989). https://doi.org/10.1007/BF00330945
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00330945