Definitions

EcoRI

EcoRI

EcoRI (pronounced "eco R one") is a nuclease enzyme isolated from certain strains of E. coli, and is part of the restriction modification system.

In molecular biology, it is a commonly used restriction enzyme. It creates sticky ends with 5' end overhangs. The nucleic acid sequence where the enzyme cuts is G|AATTC, which is a palindrome as the complementary sequence is CTTAA|G.

Structure

Primary Structure

EcoRI contains the PD..D/EXK motif within its active site like many restriction endonucleases. It is typically used in Isolation and restriction of bacterial plasmid DNA. In EcoRI this motif consists of residues P90, D91, E111, A112, K113(2).

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 (3).

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 homodimer interact with the DNA symmetrically(3). In the complex, two α-helices from each subunit come together to form a four helix bundle(2). 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

Because of their ability to cut DNA in predictable locations and leave ends which can be ligated back together, they are commonly used in cloning, DNA screening, deletion mutagenesis, and many other commonly used techniques. EcoRI is prone to exhibit star activity if excess of salts are present during the digestion.

See also

References

External links

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