EcoRI (pronounced "eco R one") is a

restriction endonuclease A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

isolated from species '' E. coli.'' It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the

restriction modification system The restriction modification system (RM system) is found in bacteria and archaea, and provides a defense against foreign DNA, such as that borne by bacteriophages. Bacteria have restriction enzymes, also called restriction endonucleases, which ...

. The ''Eco'' part of the enzyme's name originates from the species from which it was isolated"E" denotes generic name, "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. In

molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...

it is used as a

restriction enzyme A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...

. EcoRI creates 4 nucleotide

sticky ends DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends, one strand is longer than the o ...

with

5' end Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide pentose-sugar-r ...

overhangs of AATT. The nucleic acid recognition sequence where the enzyme cuts is G↓AATTC, which has a

palindromic A palindrome ( /ˈpæl.ɪn.droʊm/) is a word, number, phrase, or other sequence of symbols that reads the same backwards as forwards, such as ''madam'' or '' racecar'', the date " 02/02/2020" and the sentence: "A man, a plan, a canal – Pana ...

complementary sequence of CTTAA↓G. Other restriction enzymes, depending on their cut sites, can also leave 3' overhangs or blunt ends with no overhangs.

History

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 and

medicine Medicine is the science and Praxis (process), practice of caring for patients, managing the Medical diagnosis, diagnosis, prognosis, Preventive medicine, prevention, therapy, treatment, Palliative care, palliation of their injury or disease, ...

. EcoRI, discovered in 1970, was isolated by PhD student Robert Yoshimori who investigated clinical ''E. coli'' isolates that contained restriction systems presented on its

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...

s. The purified isolates became known as EcoRI that is used to cleave G’AATTC.

Structure

Primary structure

EcoRI contains the PD..D/EXK motif within its active site like many restriction endonucleases.

Tertiary and quaternary structure

The enzyme is a

homodimer In biochemistry, a protein dimer is a macromolecular complex or protein multimer, multimer formed by two protein monomers, or single proteins, which are usually Non-covalent interaction, non-covalently bound. Many macromolecules, such as proteins ...

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. EcoRI_restriction_enzyme_recognition_site

EcoRI_restriction_enzyme_recognition_site

EcoRI has been cocrystallized with the sequence it normally cuts. This crystal was used to solve the structure of the complex (). The solved crystal structure shows that the subunits of the enzyme

interact with the DNA symmetrically. In the complex, two α-helices from each subunit come together to form a four-helix bundle. 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.

Uses

Restriction enzymes are used in a wide variety of molecular genetics techniques including

cloning Cloning is the process of producing individual organisms with identical genomes, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction; this reproduction of an organism by itself without ...

, DNA screening and deleting sections of DNA ''in vitro''. Restriction enzymes, like EcoRI, that generate

of DNA are often used to cut DNA prior to ligation, as sticky ends make the ligation reaction more efficient. One example of this use is in

recombinant DNA Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be fo ...

production, when joining donor and vector DNA. 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.

References

External links

* * {{Portal bar, Biology, border = no EC 3.1.21 Bacterial enzymes Restriction enzymes Nucleases