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The Info List - EcoRV


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ECORV (pronounced "eco R five") is a type II restriction endonuclease isolated from certain strains of Escherichia coli . It has the alternative name Eco32I.

In molecular biology , it is a commonly used restriction enzyme . It creates blunt ends . The enzyme recognizes the palindromic 6-base DNA sequence 5'-GATATC-3' and makes a cut at the vertical line. The complementary sequence is then 3'-CTATAG-5'. The ends are blunt and can be ligated into a blunt cloning site easily but with lower efficiency than sticky ends.

CONTENTS

* 1 Structure * 2 Mode of action * 3 Uses * 4 See also * 5 References

STRUCTURE

The structure of this enzyme, and several mutants, in complex with the DNA sequence which it cuts has been solved by X-ray crystallography .

The core of the enzyme consists of a five-stranded mixed β-sheet flanked by α-helices. The core is conserved in all other type II restriction endonucleases. It also has an N-terminal dimerization subdomain formed by a short α-helix, a two-stranded antiparallel -sheet, and a long α-helix. This subdomain is found only in EcoRV
EcoRV
and PvuII.

MODE OF ACTION

Like EcoRI , EcoRV
EcoRV
forms a homodimer in solution before binding and acting on its recognition sequence . Initially the enzyme binds weakly to a non-specific site on the DNA. It randomly walks along the molecule until the specific recognition site is found. EcoRV
EcoRV
has a high specificity for its target DNA sequence.

Binding of the enzyme induces a conformational change in the DNA, bending it by about 50°. DNA bending results in the unstacking of the bases, widening of the minor groove, and compression of the major groove. This brings the phosphodiester linkage to be broken closer to the active site of the enzyme, where it can be cleaved. Cleavage occurs within the recognition sequence, and does not require ATP hydrolysis.

EcoRV
EcoRV
is the only type II restriction endonuclease known to cause a major protein-induced conformational change in the DNA.

USES

EcoRV
EcoRV
is often used to cut open a plasmid vector to insert a gene -of-interest during gene cloning . The enzyme is supplied by many manufacturers and requires bovine serum albumin to work properly.

SEE ALSO

* EcoRI , another nuclease enzyme from E. coli. * EcoRII , another nuclease enzyme from E. coli. * FokI , a nuclease enzyme from Flavobacterium okeanokoites

REFERENCES

* ^ A B C D Pingoud A, Jeltsch A (2001). "Structure and function of type II restriction endonucleases" . Nucleic Acids Research. 29 (18): 3705–3727. PMC 55916  . PMID 11557805 . doi :10.1093/nar/29.18.3705 . * ^ Bitinaite J, Wah DA, Aggarwal AK, Schildkraut I (1998). "FokI dimerization is required for DNA cleavage" . Proc Natl Acad Sci USA. 95 (18): 10570–10575. PMC 27935  . PMID 9724744 . doi :10.1073/pnas.95.18.10570 . * ^ Zahran, M., Daidone, I., Smith, J. C., ;background:none transparent;border:none;-moz-box-shadow:none;-webkit-box-shadow:none;box-shadow:none;">v

* t * e

Hydrolase : esterases (EC 3.1)

3.1.1 : Carboxylic ester hydrolases

* Cholinesterase

* Acetylcholinesterase
Acetylcholinesterase
* Butyrylcholinesterase

* Pectinesterase * 6-phosphogluconolactonase
6-phosphogluconolactonase
* PAF acetylhydrolase

* Lipase
Lipase

* Bile salt-dependent * Gastric /Lingual * Pancreatic * Lysosomal * Hormone-sensitive * Endothelial * Hepatic * Lipoprotein * Monoacylglycerol * Diacylglycerol

* Phospholipase
Phospholipase

* A1 * A2 * B

3.1.2 : THIOESTERASE

* Palmitoyl protein thioesterase * Ubiquitin carboxy-terminal hydrolase L1 * 4-hydroxybenzoyl-CoA thioesterase

3.1.3 : PHOSPHATASE

* Alkaline phosphatase

* ALPI * ALPL * ALPP

* Acid phosphatase (Prostatic )/Tartrate-resistant acid phosphatase / Purple acid phosphatases * Nucleotidase * Glucose 6-phosphatase
Glucose 6-phosphatase

* Fructose 1,6-bisphosphatase
Fructose 1,6-bisphosphatase

* Calcineurin
Calcineurin

* Protein phosphatase

* PP2A
PP2A

* OCRL
OCRL
* Pyruvate dehydrogenase phosphatase * Fructose 6-P,2-kinase:fructose 2,6-bisphosphatase * PTEN * Phytase

* Inositol-phosphate phosphatase

* IMPA1 , IMPA2 , IMPA3

* Protein phosphatase : Protein tyrosine phosphatase
Protein tyrosine phosphatase
* Protein serine/threonine phosphatase * Dual-specificity phosphatase

3.1.4 : Phosphodiesterase
Phosphodiesterase

* Autotaxin

* Phospholipase
Phospholipase

* C * D

* Sphingomyelin phosphodiesterase

* 1

* PDE1
PDE1
* PDE2 * PDE3 * PDE4A / PDE4B
PDE4B
* PDE5 * Lecithinase ( Clostridium perfringens alpha toxin ) * Cyclic nucleotide phosphodiesterase
Cyclic nucleotide phosphodiesterase

3.1.6 : SULFATASE

* arylsulfatase

* Arylsulfatase A * Arylsulfatase B * Arylsulfatase E * Steroid sulfatase
Steroid sulfatase

* Galactosamine-6 sulfatase
Galactosamine-6 sulfatase
* Iduronate-2-sulfatase * N-acetylglucosamine-6-sulfatase

Nuclease (includes deoxyribonuclease and ribonuclease )

3.1.11 -16: Exonuclease

EXODEOXYRIBONUCLEASE

* RecBCD

EXORIBONUCLEASE

* Oligonucleotidase

3.1.21 -31: Endonuclease

ENDODEOXYRIBONUCLEASE

* Deoxyribonuclease I * Deoxyribonuclease II * Deoxyribonuclease IV * Restriction enzyme
Restriction enzyme
* UvrABC endonuclease

ENDORIBONUCLEASE

* RNase III

* RNase H
RNase H

* 1 * 2A * 2B * 2C

* RNase P
RNase P

* RNase A

* 1 * 2 * 3 * 4/5

* RNase T1 * RNA-induced silencing complex
RNA-induced silencing complex

EITHER DEOXY- OR RIBO-

* Aspergillus nuclease S1 * Micrococcal nuclease
Micrococcal nuclease

* v * t * e

Enzymes

ACTIVITY

* Active site * Binding site * Catalytic triad
Catalytic triad
* Oxyanion hole
Oxyanion hole
* Enzyme
Enzyme
promiscuity * Catalytically perfect enzyme * Coenzyme
Coenzyme
* Cofactor * Enzyme
Enzyme
catalysis

REGULATION

* Allosteric regulation
Allosteric regulation
* Cooperativity
Cooperativity
* Enzyme
Enzyme
inhibitor

CLASSIFICATION

* EC number * Enzyme
Enzyme
superfamily * Enzyme
Enzyme
family * List of enzymes

KINETICS

* Enzyme
Enzyme
kinetics * Eadie–Hofstee diagram * Hanes–Woolf plot * Lineweaver–Burk plot
Lineweaver–Burk plot
* Michaelis–Menten kinetics

TYPES

* EC1 OXIDOREDUCTASES (list ) * EC2 TRANSFERASES (list ) * EC3 HYDROLASES (list ) * EC4 LYASES (list ) * EC5 ISOMERASES (list ) * EC6 LIGASES (list )

* Molecular and Cellular Biology portal

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EcoRV
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