''Eco''RI (pronounced "eco R one") is a restriction

endonuclease Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...

enzyme isolated from species ''

E. coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...

.'' 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 other prokaryotic organisms, and provides a defense against foreign DNA, such as that borne by bacteriophages. Bacteria have restriction enzymes, also called restriction end ...

. The ''Eco'' part 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. In

molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...

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 ...

. ''Eco''RI 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 ...

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-ri ...

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

palindromic A palindrome is a word, number, phrase, or other sequence of symbols that reads the same backwards as forwards, such as the words ''madam'' or ''racecar'', the date and time ''11/11/11 11:11,'' 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.

Structure

Primary structure

''Eco''RI contains the PD..D/EXK motif within its active site like many

restriction endonucleases 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 ...

Tertiary and quaternary structure

The enzyme is a

homodimer In biochemistry, a protein dimer is a macromolecular complex formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ''dimer'' ha ...

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

''Eco''RI 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 The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...

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 or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, cl ...

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

of DNA are often used to cut DNA prior to

ligation Ligation may refer to: * Ligation (molecular biology), the covalent linking of two ends of DNA or RNA molecules * In medicine, the making of a ligature (tie) * Chemical ligation, the production of peptides from amino acids * Tubal ligation, a meth ...

, 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 foun ...

production, when joining donor and vector DNA. ''Eco''RI can exhibit non-site-specific cutting, known as

star activity Star activity is the relaxation or alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under reaction conditions that differ significantly from those optimal for the enzyme. The result is typically cleavage a ...

, depending on the conditions present in the reaction. Conditions that can induce star activity when using ''Eco''RI 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