Current Genetics

, Volume 49, Issue 5, pp 333–340 | Cite as

Chlamydomonas reinhardtii encodes a single sigma70-like factor which likely functions in chloroplast transcription

  • Alexandra-V. Bohne
  • Vered Irihimovitch
  • Andreas Weihe
  • David B. Stern
Research Article

Abstract

Chlamydomonas reinhardtii EST clones encoding a protein highly similar to prokaryotic sigma factors and plant sigma-like factors (SLFs) were used to isolate a BAC clone containing the full-length gene CrRpoD. The gene is likely to be single-copy, in contrast to small gene families encoding SLFs in plants. The CrRpoD mRNA comprises 3,033 nt with an open reading frame of 2,256 nt, encoding a putative protein of 752 amino acids with a molecular mass of 80.2 kDa. The sequence contains conserved regions 2–4 typically found in sigma factors, and an unusually long amino terminal extension, which by in silico analysis has properties of a chloroplast transit peptide. Expression of CrRpoD was confirmed by immunodetection of a 85 kDa polypeptide in a preparation enriched for chloroplast proteins. To demonstrate functionality in transcription initiation, a recombinant CrRpoD–thioredoxin fusion protein was reconstituted with E. coli RNA polymerase core enzyme and tested in vitro. This chimeric holoenzyme specifically bound the spinach psbA and Chlamydomonas rrn16 promoters in gel mobility shift assays and exhibited specific transcription initiation from the same two promoters, providing evidence for the role of CrRpoD as a functional transcription factor.

Keywords

Organelle transcription In vitro transcription Chloroplast RNA polymerase Green algae 

Supplementary material

294_2006_60_MOESM1_ESM.pdf (3.1 mb)
Supplementary material

References

  1. Armstrong N, de Lencastre A, Gouaux E (1999) A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase. Protein Sci 8:1475–1483PubMedGoogle Scholar
  2. Beardslee TA, Roy-Chowdhury S, Jaiswal P, Buhot L, Lerbs-Mache S, Stern DB, Allison LA (2002) A nucleus-encoded maize protein with sigma factor activity accumulates in mitochondria and chloroplasts. Plant J 31:199–209PubMedCrossRefGoogle Scholar
  3. Brahamsha B, Haselkorn R (1991) Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 173:2442–2450PubMedGoogle Scholar
  4. Brahamsha B, Haselkorn R (1992) Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp. strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes. J Bacteriol 174:7273–7282PubMedGoogle Scholar
  5. Burton Z, Burgess RR, Lin J, Moore D, Holder S, Gross CA (1981) The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit from E. coli K12. Nucleic Acids Res 9:2889–2903PubMedCrossRefGoogle Scholar
  6. Carter ML, Smith AC, Kobayashi H, Purton S, Herrin DL (2004) Structure, circadian regulation and bioinformatic analysis of the unique sigma factor gene in Chlamydomonas reinhardtii. Photosynth Res 82:339–349PubMedCrossRefGoogle Scholar
  7. Eberhard S, Drapier D, Wollman FA (2002) Searching limiting steps in the expression of chloroplast-encoded proteins: relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii. Plant J 31:149–160PubMedCrossRefGoogle Scholar
  8. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016PubMedCrossRefGoogle Scholar
  9. Fischer N, Stampacchia O, Redding K, Rochaix JD (1996) Selectable marker recycling in the chloroplast. Mol Gen Genet 251:373–380PubMedCrossRefGoogle Scholar
  10. Fujiwara M, Nagashima A, Kanamaru K, Tanaka K, Takahashi H (2000) Three new nuclear genes, sigD, sigE and sigF, encoding putative plastid RNA polymerase sigma factors in Arabidopsis thaliana. FEBS Lett 481:47–52PubMedCrossRefGoogle Scholar
  11. Gitt MA, Wang LF, Doi RH (1985) A strong sequence homology exists between the major RNA polymerase sigma factors of Bacillus subtilis and Escherichia coli. J Biol Chem 260:7178–7185PubMedGoogle Scholar
  12. Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation of Chlamydomonas. Nucleic Acids Res 19:4083–4090PubMedCrossRefGoogle Scholar
  13. Goldschmidt-Clermont M, Malnoe P, Rochaix J-D (1989) Preparation of Chlamydomonas chloroplasts for the in vitro import of polypeptide precursors. Plant Physiol 89:15–18PubMedGoogle Scholar
  14. Hakimi MA, Privat I, Valay JG, Lerbs-Mache S (2000) Evolutionary conservation of C-terminal domains of primary sigma(70)-type transcription factors between plants and bacteria. J Biol Chem 275:9215–9221PubMedCrossRefGoogle Scholar
  15. Hanaoka M, Kanamaru K, Takahashi H, Tanaka K (2003) Molecular genetic analysis of chloroplast gene promoters dependent on SIG2, a nucleus-encoded sigma factor for the plastid-encoded RNA polymerase, in Arabidopsis thaliana. Nucleic Acids Res 31:7090–7098PubMedCrossRefGoogle Scholar
  16. Harris EH (1989) The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. Academic, San DiegoGoogle Scholar
  17. Hess WR, Boerner T (1999) Organellar RNA polymerases of higher plants. Int Rev Cytol 190:1–59PubMedCrossRefGoogle Scholar
  18. Homann A, Link G (2003) DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression. Eur J Biochem 270:1288–1300PubMedCrossRefGoogle Scholar
  19. Isono K et al (1997) Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of sigma-70 factors of bacterial RNA polymerases in Arabidopsis thaliana. Proc Natl Acad Sci USA 94:14948–14953PubMedCrossRefGoogle Scholar
  20. Kanamaru K et al (1999) Plastidic RNA polymerase sigma factors in Arabidopsis. Plant Cell Physiol 40:832–842PubMedGoogle Scholar
  21. Kestermann M, Neukirchen S, Kloppstech K, Link G (1998) Sequence and expression characteristics of a nuclear-encoded chloroplast sigma factor from mustard (Sinapis alba). Nucleic Acids Res 26:2747–2753PubMedCrossRefGoogle Scholar
  22. Klein U, De Camp JD, Bogorad L (1992) Two types of chloroplast gene promoters in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 89:3453–3457PubMedCrossRefGoogle Scholar
  23. Klinkert B, Schwarz C, Pohlmann S, Pierre Y, Girard-Bascou J, Nickelsen J (2005) Relationship between mRNA levels and protein accumulation in a chloroplast promoter-mutant of Chlamydomonas reinhardtii. Mol Genet Genomics 274:637–643PubMedCrossRefGoogle Scholar
  24. Lahiri SD, Allison LA (2000) Complementary expression of two plastid-localized sigma-like factors in maize. Plant Physiol 123:883–894PubMedCrossRefGoogle Scholar
  25. Lahiri SD, Yao J, McCumbers C, Allison LA (1999) Tissue-specific and light-dependent expression within a family of nuclear-encoded sigma-like factors from Zea mays. Mol Cell Biol Res Commun 1:14–20PubMedCrossRefGoogle Scholar
  26. Liere K, Maliga P (2001) Plastid RNA polymerases in higher plants. In: Andersson B, Aro EM (eds) Regulation of photosynthesis. Kluwer, Rotterdam, pp 29–39Google Scholar
  27. Liu B, Troxler RF (1996) Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium. Proc Natl Acad Sci USA 93:3313–3318PubMedCrossRefGoogle Scholar
  28. Maul JE, Lilly JW, Cui L, dePamphilis CW, Miller W, Harris EH, Stern DB (2002) The Chlamydomonas reinhardtii plastid chromosome: islands of genes in a sea of repeats. Plant Cell 14:2659–2679PubMedCrossRefGoogle Scholar
  29. Morikawa K, Ito S, Tsunoyama Y, Nakahira Y, Shiina T, Toyoshima Y (1999) Circadian-regulated expression of a nuclear-encoded plastid sigma factor gene (sigA) in wheat seedlings. FEBS Lett 451:275–278PubMedCrossRefGoogle Scholar
  30. Morikawa K, Shiina T, Murakami S, Toyoshima Y (2002) Novel nuclear-encoded proteins interacting with a plastid sigma factor, Sig1, in Arabidopsis thaliana. FEBS Lett 514:300–304PubMedCrossRefGoogle Scholar
  31. Oikawa K, Tanaka K, Takahashi H (1998) Two types of differentially photo-regulated nuclear genes that encode sigma factors for chloroplast RNA polymerase in the red alga Cyanidium caldarium strain RK-1. Gene 210:277–285PubMedCrossRefGoogle Scholar
  32. Oikawa K, Fujiwara M, Nakazato E, Tanaka K, Takahashi H (2000) Characterization of two plastid sigma factors, SigA1 and SigA2, that mainly function in matured chloroplasts in Nicotiana tabacum. Gene 261:221–228PubMedCrossRefGoogle Scholar
  33. Rochaix JD (1995) Chlamydomonas reinhardtii as the photosynthetic yeast. Annu Rev Genet 29:209–230PubMedCrossRefGoogle Scholar
  34. 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 51:1145–1157PubMedCrossRefGoogle Scholar
  35. Surzycki SJ, Shellenbarger DL (1976) Purification and characterization of a putative sigma factor from Chalamydomonas reinhardii. Proc Natl Acad Sci USA 73:3961–3965PubMedCrossRefGoogle Scholar
  36. Tan S, Troxler RF (1999) Characterization of two chloroplast RNA polymerase sigma factors from Zea mays: photoregulation and differential expression. Proc Natl Acad Sci USA 96:5316–5321PubMedCrossRefGoogle Scholar
  37. Tanaka K et al (1997) Characterization of three cDNA species encoding plastid RNA polymerase sigma factors in Arabidopsis thaliana: evidence for the sigma factor heterogeneity in higher plant plastids. FEBS Lett 413:309–313PubMedCrossRefGoogle Scholar
  38. Tozawa Y, Tanaka K, Takahashi H, Wakasa K (1998) Nuclear encoding of a plastid sigma factor in rice and its tissue- and light-dependent expression. Nucleic Acids Res 26:415–419PubMedCrossRefGoogle Scholar
  39. Yao J, Roy-Chowdhury S, Allison LA (2003) AtSig5 is an essential nucleus-encoded Arabidopsis sigma-like factor. Plant Physiol 132:739–747PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Alexandra-V. Bohne
    • 1
    • 2
  • Vered Irihimovitch
    • 2
  • Andreas Weihe
    • 1
  • David B. Stern
    • 2
  1. 1.Institut für BiologieHumboldt-UniversitätBerlinGermany
  2. 2.Boyce Thompson Institute for Plant ResearchCornell UniversityIthacaUSA

Personalised recommendations