葉緑体ゲノム装置の不連続進化仮説 - PowerPoint PPT Presentation

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葉緑体ゲノム装置の不連続進化仮説

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  1. 葉緑体ゲノム装置の不連続進化仮説

  2. 葉緑体ゲノム装置の構造と進化 • 葉緑体ゲノムとゲノム装置 • 複製酵素,転写酵素,DNA結合タンパク質,RNA結合タンパク質 • 比較ゲノム学によるゲノム装置成分の検索 • 核様体の比較生化学 • ゲノム装置の不連続進化

  3. シアノバクテリアと植物・藻類の葉緑体およびそれらの核様体シアノバクテリアと植物・藻類の葉緑体およびそれらの核様体

  4. 葉緑体ゲノム装置の起源の探索 • 複製:DNA polymerase(s) • 転写:ファージ型RNA polymerase(s) • DNA結合タンパク質(転写因子): • HU, DnaB helicase • SiR (亜硫酸還元酵素) • PEND(包膜のDNA結合タンパク質) • RNA結合タンパク質:Rbp / GRP

  5. Discontinuous evolution of plastid genomic machinery(1) DNA polymerase Adapted from N. Sato (2001) Trends in Plant Science 6: 151-156


  6. Comparison of various nucleoids

  7. Lack of prokaryotic DNA-binding proteins in plastids – comparative genomics cp, chloroplast genome; nuc, nuclear genome. The Cyanidioschyzon data were used as the ‘nuc’ data for Rhodo- and Chromophytes.

  8. Presence of HU protein in cyanobacteria and rhodophyte plastids

  9. Sulfite reductase is a major protein in plastid nucleoid

  10. Formation of particulate complex of purified sulfite reductase with cpDNA Water control BSA control + cpDNA (2 hr) + cpDNA (24 hr)

  11. Models of sulfite reductases E. coli Maize

  12. 転写活性に対するヘパリンとSiRの効果 Approximately, 10 sugar residues of heparin counteract with the action of one SiR molecule

  13. SiR is also present in isolated moss nucleoids

  14. Alignment of sulfite reductases of plants and cyanobacteria

  15. HUとSiRの機能的比較 SiR HU

  16. DNA-binding proteins of plastids reported in the literature

  17. 紅藻と植物の DNA polymerases

  18. 植物オルガネラ型DNA polymerasesの系統

  19. 葉緑体とミトコンドリアの複製系の起源 植物・藻類では,葉緑体とミトコンドリアの複製系は極めて似ており,同一のDNA polymeraseが働いている。 しかしこのDNA polymeraseの起源は,シアノバクテリアやαプロテオバクテリアに求めることはできない。 動物や菌類と植物・藻類では,ミトコンドリアDNA polymeraseが異なっている。

  20. Origin of NEPRecent evolution of T7-like RNA polymerases

  21. Origin of NEP In angiosperm chloroplasts, two types of RNA polymerases (RNAP) are present: one is a prokaryotic RNAP called PEP, which is encoded in the chloroplast genome, while another is a phage-type RNAP called NEP, which is encoded in the nuclear genome. The phage-type RNAP consists of a single polypeptide, and functions in mitochondria of most eukaryotes including yeast and human. We analyzed the phage-type RNAP in the moss Physcomitrella patens.

  22. Model of organellar RNA polymerases in in higher plants

  23. Two cDNAs: RpoT1 and RpoT2

  24. Expression and purification of PpRPOT proteins Nuclear- encoded phage-type RNA polymerase of Physcomitrella patens (moss)

  25. Enzymatic activity of the T7-type RNA polymerases in Physcomitrella 60 ng/ml 30 ng/ ml 2 mg 80 ng/ml 40 ng/ ml

  26. Mitochondrial targeting of the PpRPOT proteins

  27. Phylogeny of T7-type RNA polymerases

  28. Phylogeny of T7-type RNA polymerases(2)

  29. Signature sequence

  30. NEP-dependent promoters of chloroplast genes might be specific to angiosperms NCII : Non-consensus type promoter II, which is transcribed by NEP.

  31. Recent origin of NEP (2) The NEP, a nuclear-encoded phage-type RNA polymerase of chloroplasts, has been created by duplication of the gene encoding a mitochondrial counterpart. Phylogenetic analysis of the polymerases as well as the structure of NEP-dependent promoters suggest that this gene duplication occurred after the evolution of angiosperms. Plant & Cell Physiology 43: 245-255 (2002)

  32. Conflicting results on the targeting of the two RNAPs of Physcomitrella patens • Kabeya and Sato (2002) Plant Cell Physiol. 43: 245-255 • Targeting to mitochondria (no targeting to chloroplasts) • Richter et al. (2002) Gene 290: 95-105 • Dual targeting (mostly to chloroplasts)

  33. Chloroplast targeting ?? Forced translation from the first AUG

  34. Mitochondrial targeting of the PpRPOT proteins (2) Translation within the natural context

  35. Two methionine codons

  36. GFP in transient expression

  37. Stable transformants

  38. Immunoblot

  39. Tagetitoxin sensitivity

  40. Translation efficiency

  41. コケでも被子植物でも これまで他のグループによって2重ターゲティングが提唱されていた,細胞核にコードされたRNA polymerase (RPOT)は,いずれもミトコンドリアだけにターゲティングされること,その理由は,本来の5’ UTRコンテキストでは,2番目のメチオニンコドンだけからしか翻訳されないためであること,が判明しいた。 従って,本来の5’ UTRを持たない人工的に作られたGFP融合タンパク質に基づくターゲティングは,正しい結果をもたらさないということが教訓である。

  42. Dually targeted DNA-binding protein, PEND

  43. 若いエンドウの葉では,核様体が包膜に結合している若いエンドウの葉では,核様体が包膜に結合している 播種後6日目 播種後14日目 5 µm

  44. 核様体の包膜結合に関与するDNA結合タンパク質PEND protein: Sato et al. (1993) EMBO J. 12: 555-561.

  45. Import of the PEND protein

  46. Import of the PEND protein (2)

  47. Import of the PEND protein (3) Localization of the full-length protein to the chloroplast envelope

  48. Dual targeting of the PEND protein Initial translation product is targeted to plastid envelope. The N-terminus is processed. The C-terminus is involved in membrane-binding. If the N-terminal half of the mature PEND protein is cleaved, this polypeptide may be re-targeted to the nucleus. BnGSBF1, a PEND homolog, is supposed to act as a transcription regulator in CAB gene.

  49. Selection of binding sites for the PEND protein (cbZIP region)

  50. Binding sites for the PEND protein in the pea cpDNA