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出典(authority):フリー百科事典『ウィキペディア(Wikipedia)』「2018/02/15 19:20:34」(JST)
[Wiki ja表示]
EcoRI(えこあーるわん)は、II型の制限酵素の一種で、制限酵素としては最も代表的なものである。
目次
- 1 歴史
- 2 作用
- 3 構造
- 4 関連項目
- 5 脚注
歴史
1972年、カリフォルニア大学のハーバート・ボイヤーの研究室で大腸菌から単離された。大腸菌(Escherichia coli)のRY13株から見つかった1番目の制限酵素ということで、「EcoRI」と命名された[1]。
1986年、制限酵素として初めて結晶構造が報告された[2]。
作用
EcoRIはIIP型に分類される制限酵素であり、DNA中の 5'-GAATTC-3' というパリンドロームになっている6塩基配列を認識し、GとAの間に切れ目を入れ、切り口に付着末端を作り出す。この時、酵素との結合部でDNAを直角に屈曲させることが分かっている。なお、大腸菌自身の遺伝子は、この配列の部分がメチル化されているため、切断される心配はない。
EcoRIの切断パターン
切断前の配列 切断後の配列
5'-GAATTC-3' 5'-G AATTC-3'
3'-CTTAAG-5' 3'-CTTAA G-5'
構造
一次構造
他の多くの制限酵素と同様に活性部位にPD...D/ExKモチーフを持つ[2]。
三次・四次構造
約31kDaのサブユニットがホモ2量体を形成し、α/β構造である球状ドメインでDNAと結合する。
球状ドメインから飛び出しているループ領域はDNAを包むように結合し、認識配列の外のDNA骨格と結合する。この結合が、認識配列近傍のDNA配列がEcoRIの活性に影響を与えている原因であると考えられている。
それぞれのサブユニットの2つのαヘリックスは4ヘリックスバンドルを構成し、DNAの主溝に入り込み結合する。βストランドにはヌクレアーゼ活性に必要なアミノ酸残基が含まれている[2]。
関連項目
脚注
- ^ Dubey, R. C. (2014). Advanced Biotechnology. S. Chand Publishing. p. 259. ISBN 9788121942904.
- ^ a b c Alfred Pingoud; Geoffrey G. Wilson; Wolfgang Wende (2014). “Type II restriction endonucleases—a historical perspective and more”. Nucleic Acids Research 42 (12): 7489-7527. https://doi.org/10.1093/nar/gku447.
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この項目は、生物学に関連した書きかけの項目です。この項目を加筆・訂正などしてくださる協力者を求めています(プロジェクト:生命科学/Portal:生物学)。 |
[Wiki en表示]
EcoRI |
The crystallographic structure of restriction endonuclease EcoRI at 3.3 a in the absence of DNA
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Identifiers |
Symbol |
EcoRI |
Pfam |
PF02963 |
InterPro |
IPR004221 |
SCOP |
1na6 |
SUPERFAMILY |
1na6 |
Available protein structures: |
Pfam |
structures |
PDB |
RCSB PDB; PDBe; PDBj |
PDBsum |
structure summary |
|
EcoRI (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species E. coli. The Eco part of the enzyme's name originates from the species from which it was isolated, while the R represents the particular strain, in this case RY13. The last part of its name, the I, denotes that it was the first enzyme isolated from this strain. EcoRI is a restriction enzyme that cleaves DNA double helixes into fragments at specific sites. It is also a part of the restriction modification system.
In molecular biology it is used as a restriction enzyme. EcoRI creates 4 nucleotide sticky ends with 5' end overhangs of AATT. The nucleic acid recognition sequence where the enzyme cuts is G/AATTC, which has a palindromic, complementary sequence of CTTAA/G. The / in the sequence indicates which phosphodiester bond the enzyme will break in the DNA molecule. Other restriction enzymes, depending on their cut sites, can also leave 3' overhangs or blunt ends with no overhangs.
Contents
- 1 Structure
- 1.1 Primary structure
- 1.2 Tertiary and quaternary structure
- 2 Uses
- 3 See also
- 4 References
- 5 External links
Structure
Primary structure
EcoRI contains the PD..D/EXK motif within its active site like many restriction endonucleases.
EcoRI crystal structure. Dimer bound to DNA (PDB
1ckq)
Tertiary and quaternary structure
The enzyme is a homodimer of a 31 kilodalton subunit consisting of one globular domain of the α/β architecture. Each subunit contains a loop which sticks out from the globular domain and wraps around the DNA when bound.[1][2]
EcoRI recognition site with cutting pattern indicated by a green line
EcoRI has been cocrystallized with the sequence it normally cuts. This crystal was used to solve the structure of the complex 1QPS. The solved crystal structure shows that the subunits of the enzyme homodimer interact with the DNA symmetrically.[1] In the complex, two α-helices from each subunit come together to form a four-helix bundle.[3] On the interacting helices are residues Glu144 and Arg145, which interact together, forming a crosstalk ring that is believed to allow the enzyme's two active sites to communicate.[4]
Uses
Restriction enzymes, such as EcoRI, are used in a wide variety of molecular genetics techniques including cloning, DNA screening and deleting sections of DNA in vitro. Restriction enzymes, like EcoRI, that generate sticky ends of DNA are often used to cut DNA prior to ligation, as the sticky ends make the ligation reaction more efficient. EcoRI can exhibit non-site-specific cutting, known as star activity, depending on the conditions present in the reaction. Conditions that can induce star activity when using EcoRI include low salt concentration, high glycerol concentration, excessive amounts of enzyme present in the reaction, high pH and contamination with certain organic solvents.[5]
See also
- EcoRII, another nuclease enzyme from E. coli.
- EcoRV, another nuclease enzyme from E. coli.
References
- ^ a b Pingoud, A.; Jeltsch, A. (2001). "Structure function of type II restriction endonucleases". Nucleic Acids Res. 29 (18): 3705–3727. doi:10.1093/nar/29.18.3705. PMC 55916 . PMID 11557805.
- ^ Kurpiewski, M. R.; Engler, L. E.; Wozniak, L. A.; Kobylanska, A.; Koziolkiewicz, M.; Stec, W. J; Jen-Jacobson, L (2004). "Mechanisms of coupling between DNA recognition catalysis in EcoRI endonucleases". Structure. 12: 1775–1788. doi:10.1016/j.str.2004.07.016. PMID 15458627.
- ^ Bitinaite, J. D. A.; Wah Aggarwal, A. K.; Schildkraut, I. (1998). "FokI dimerization is required for DNA cleavage". Proc Natl Acad Sci U S A. 95 (18): 10570–5. doi:10.1073/pnas.95.18.10570. PMC 27935 . PMID 9724744.
- ^ Kim, Y. C.; Grable, J. C.; Love, R.; Greene, P. J.; Rosenberg, J. M. (1990). "Refinement of EcoRI endonuclease crystal structure: a revised protein chain tracing". Science. 249 (4974): 1307–1309. doi:10.1126/science.2399465. PMID 2399465.
- ^ http://www.neb.com/nebecomm/products/faqproductR0101.asp#1
External links
- Eco-RI at the US National Library of Medicine Medical Subject Headings (MeSH)
Hydrolase: esterases (EC 3.1)
|
3.1.1: Carboxylic
ester hydrolases |
- Cholinesterase
- Acetylcholinesterase
- Butyrylcholinesterase
- Pectinesterase
- 6-phosphogluconolactonase
- PAF acetylhydrolase
- Lipase
- Bile salt-dependent
- Gastric/Lingual
- Pancreatic
- Lysosomal
- Hormone-sensitive
- Endothelial
- Hepatic
- Lipoprotein
- Monoacylglycerol
- Diacylglycerol
|
3.1.2: Thioesterase |
- Palmitoyl protein thioesterase
- Ubiquitin carboxy-terminal hydrolase L1
- 4-hydroxybenzoyl-CoA thioesterase
|
3.1.3: Phosphatase |
- Alkaline phosphatase
- Acid phosphatase (Prostatic)/Tartrate-resistant acid phosphatase/Purple acid phosphatases
- Nucleotidase
- Glucose 6-phosphatase
- Fructose 1,6-bisphosphatase
- Protein phosphatase
- OCRL
- Pyruvate dehydrogenase phosphatase
- Fructose 6-P,2-kinase:fructose 2,6-bisphosphatase
- PTEN
- Phytase
- Inositol-phosphate phosphatase
- Protein phosphatase: Protein tyrosine phosphatase
- Protein serine/threonine phosphatase
- Dual-specificity phosphatase
|
3.1.4:
Phosphodiesterase |
- Autotaxin
- Phospholipase
- Sphingomyelin phosphodiesterase
- PDE1
- PDE2
- PDE3
- PDE4A/PDE4B
- PDE5
- Lecithinase (Clostridium perfringens alpha toxin)
- Cyclic nucleotide phosphodiesterase
|
3.1.6: Sulfatase |
- arylsulfatase
- Arylsulfatase A
- Arylsulfatase B
- Arylsulfatase E
- Steroid sulfatase
- Galactosamine-6 sulfatase
- Iduronate-2-sulfatase
- N-acetylglucosamine-6-sulfatase
|
Nuclease (includes
deoxyribonuclease
and ribonuclease) |
3.1.11-16:
Exonuclease |
Exodeoxyribonuclease |
|
Exoribonuclease |
|
|
3.1.21-31:
Endonuclease |
Endodeoxyribonuclease |
- Deoxyribonuclease I
- Deoxyribonuclease II
- Deoxyribonuclease IV
- Restriction enzyme
- UvrABC endonuclease
|
Endoribonuclease |
- RNase III
- RNase H
- RNase P
- RNase A
- RNase T1
- RNA-induced silencing complex
|
either deoxy- or ribo- |
- Aspergillus nuclease S1
- Micrococcal nuclease
|
|
|
Enzymes
|
Activity |
- Active site
- Binding site
- Catalytic triad
- Oxyanion hole
- Enzyme promiscuity
- Catalytically perfect enzyme
- Coenzyme
- Cofactor
- Enzyme catalysis
|
Regulation |
- Allosteric regulation
- Cooperativity
- Enzyme inhibitor
|
Classification |
- EC number
- Enzyme superfamily
- Enzyme family
- List of enzymes
|
Kinetics |
- Enzyme kinetics
- Eadie–Hofstee diagram
- Hanes–Woolf plot
- Lineweaver–Burk plot
- Michaelis–Menten kinetics
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Types |
- EC1 Oxidoreductases (list)
- EC2 Transferases (list)
- EC3 Hydrolases (list)
- EC4 Lyases (list)
- EC5 Isomerases (list)
- EC6 Ligases (list)
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UpToDate Contents
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…100. In most patients with FSHD, one D4Z4 allele is contracted (1 to 10 repeat units; EcoRI fragments <38 kb; EcoRI/BlnI fragments <35 kb), and the other D4Z4 allele has the normal number (11 to 100 repeat …
English Journal
- Engineered selective plant male sterility through pollen-specific expression of the EcoRI restriction endonuclease.
- Millwood RJ1, Moon HS1, Poovaiah CR1, Muthukumar B1, Rice JH1, Abercrombie JM1, Abercrombie LL1, Green WD1, Stewart CN Jr1.
- Plant biotechnology journal.Plant Biotechnol J.2016 May;14(5):1281-90. doi: 10.1111/pbi.12493. Epub 2015 Oct 26.
- Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some
- PMID 26503160
- CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening to enhance genome editing.
- Chiang TW1,2, le Sage C1, Larrieu D1, Demir M1, Jackson SP1,2,3.
- Scientific reports.Sci Rep.2016 Apr 15;6:24356. doi: 10.1038/srep24356.
- The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system's specificity. Recently a "double-nicking" strategy using catalytic mutant Cas9(D10
- PMID 27079678
Japanese Journal
- Genotyping and Chlorine-Resistance of Methylobacterium aquaticum Isolated from Water Samples in Japan
- FURUHATA KATSUNORI,BANZAI AZUSA U.,KAWAKAMI YASUSHI,ISHIZAKI NAOTO,YOSHIDA YOSHIHIRO,GOTO KEIICHI,FUKUYAMA MASAFUMI
- Biocontrol science 16(3), 103-107, 2011-09-10
- … The ribopatterns after digestion with EcoRI obtained from 14 strains of M. …
- NAID 10029767017
Related Links
- DNA塩基配列から各制限酵素認識配列を検索することができる簡単便利なWebツール【 Takara Cut-Site Navigator 】を公開しました。 制限酵素を使用される際に、ぜひ一度お試しください。
- 特徴 毒物扱いが解除されました(2013年2月12日出荷分より) 詳細はこちら 現在 ハイフィデリティー(HF)バージョン も販売中です。 性能を向上しつつも、同価格で提供している EcoRI-HF のご使用をお勧めします!
- EcoRI (high conc.) 314-01751 12,000 units 9,000円 高濃度品 製造元 (株)ニッポンジーン 表示価格は希望納入価格 (税別) です。 製品説明 製品内容 使用例 Q & A 資料 Data Sheet 関連情報 問い合わせ先 製品説明 制限酵素は ...
Related Pictures
★リンクテーブル★
[★]
- 英
- restriction enzyme EcoRI、deoxyribonuclease EcoRI
[★]
制限酵素EcoRI
- 関
- restriction enzyme EcoRI
[★]
制限酵素EcoRI
- 関
- deoxyribonuclease EcoRI