EcoRI
EcoRI | |||||||||
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Identifiers | |||||||||
Symbol | EcoRI | ||||||||
Pfam | PF02963 | ||||||||
InterPro | IPR004221 | ||||||||
SCOP2 | 1na6/SCOPe/SUPFAM | ||||||||
CDD | 79lll | ||||||||
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EcoRI(pronounced "eco R one" ) is arestriction endonucleaseenzymeisolated from speciesE. coli.It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of therestriction modification system.[1]TheEcopart of the enzyme's name originates from the species from which it was isolated - "E" denotes generic name which is "Escherichia" and "co" denotes species name, "coli" - while the R represents the particular strain, in this case RY13, and the I denotes that it was the first enzyme isolated from this strain.[citation needed]
Inmolecular biologyit is used as arestriction enzyme.EcoRI creates 4 nucleotidesticky endswith5' endoverhangs of AATT. The nucleic acid recognition sequence where the enzyme cuts is G↓AATTC, which has apalindromiccomplementary sequence of CTTAA↓G.[2]Other restriction enzymes, depending on their cut sites, can also leave 3' overhangs or blunt ends with no overhangs.
History
[edit]EcoRI is an example of type II restriction enzymes which now has more the 300 enzymes with more than 200 different sequence-specificities, which has transformed molecular biology andmedicine.[3]
EcoRI, discovered in 1970, was isolated by PhD student Robert Yoshimori who investigated clinicalE. coliisolates that contained restriction systems presented on itsplasmids.[3]The purified isolates became known as EcoRI that is used to cleave G’AATTC.[2]
Structure
[edit]Primary structure
[edit]EcoRI contains the PD..D/EXK motif within its active site like manyrestriction endonucleases.
Tertiary and quaternary structure
[edit]The enzyme is ahomodimerof 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.[4][5]
![](https://upload.wikimedia.org/wikipedia/commons/thumb/7/78/EcoRI_restriction_enzyme_recognition_site.svg/175px-EcoRI_restriction_enzyme_recognition_site.svg.png)
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 enzymehomodimerinteract with the DNA symmetrically.[4]In the complex, twoα-helicesfrom each subunit come together to form a four-helix bundle.[6]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.[7]
Uses
[edit]Restriction enzymes are used in a wide variety of molecular genetics techniques includingcloning,DNA screening and deleting sections of DNAin vitro.Restriction enzymes, likeEcoRI, that generatesticky endsof DNA are often used to cut DNA prior toligation,as sticky ends make theligation reactionmore efficient.[8]One example of this use is inrecombinant DNAproduction, when joining donor and vector DNA.[9]EcoRI can exhibit non-site-specific cutting, known asstar activity,depending on the conditions present in the reaction. Conditions that can induce star activity when usingEcoRI include low salt concentration, high glycerol concentration, excessive amounts of enzyme present in the reaction, high pH and contamination with certain organic solvents.[10]
See also
[edit]References
[edit]- ^Halford, S. E.; Johnson, N. P. (1980-11-01)."The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies".The Biochemical Journal.191(2): 593–604.doi:10.1042/bj1910593.ISSN0264-6021.PMC1162251.PMID6263250.
- ^abNevinsky, Georgy A. (2021-01-29)."How Enzymes, Proteins, and Antibodies Recognize Extended DNAs; General Regularities".International Journal of Molecular Sciences.22(3): 1369.doi:10.3390/ijms22031369.ISSN1422-0067.PMC7866405.PMID33573045.
- ^abLoenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.; Wilson, Geoffrey G.; Murray, Noreen E. (January 2014)."Highlights of the DNA cutters: a short history of the restriction enzymes".Nucleic Acids Research.42(1): 3–19.doi:10.1093/nar/gkt990.ISSN1362-4962.PMC3874209.PMID24141096.
- ^abPingoud A, Jeltsch A (September 2001)."Structure and function of type II restriction endonucleases".Nucleic Acids Research.29(18): 3705–27.doi:10.1093/nar/29.18.3705.PMC55916.PMID11557805.
- ^Kurpiewski MR, Engler LE, Wozniak LA, Kobylanska A, Koziolkiewicz M, Stec WJ, Jen-Jacobson L (October 2004)."Mechanisms of coupling between DNA recognition specificity and catalysis in EcoRI endonuclease".Structure.12(10): 1775–88.doi:10.1016/j.str.2004.07.016.PMID15458627.
- ^Bitinaite J, Wah DA, Aggarwal AK, Schildkraut I (September 1998)."FokI dimerization is required for DNA cleavage".Proceedings of the National Academy of Sciences of the United States of America.95(18): 10570–5.Bibcode:1998PNAS...9510570B.doi:10.1073/pnas.95.18.10570.PMC27935.PMID9724744.
- ^Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM (September 1990). "Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing".Science.249(4974): 1307–9.Bibcode:1990Sci...249.1307K.doi:10.1126/science.2399465.PMID2399465.
- ^Gao, T; Konomura, S; May, C; Nich, C (April 2015)."Increasing Overhang GC-Content Increases Sticky-End Ligation Efficiency"(PDF).Journal of Experimental Microbiology and Immunology.
- ^Griffiths, Anthony JF; Miller, Jeffrey H.; Suzuki, David T.; Lewontin, Richard C.; Gelbart, William M. (2000)."Making recombinant DNA".An Introduction to Genetic Analysis. 7th Edition.
- ^"FAQs for EcoRI, Restriction Endonucleases, NEB".Archived fromthe originalon 2012-10-15.Retrieved2010-01-21.