The crystallographic structure of restriction endonuclease EcoRI at 3.3 a in the absence of DNA
In molecular biology it is used as a restriction enzyme. It creates 4 nucleotide sticky ends with 5' end overhangs of AATT. The nucleic acid sequence where the enzyme cuts is GAATTC, which, as the complementary sequence is CTTAAG, has rotational symmetry.
EcoRI contains the PD..D/EXK motif within its active site like many restriction endonucleases.
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.
EcoRI has been cocrystallized with the sequence it normally cuts. This crystal was used to solve the structure of the complex homodimer 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.. The solved crystal structure shows that the subunits of the enzyme
Restriction enzymes such as EcoRI are used in a wide variety of molecular genetics techniques including cloning, DNA screening and deleting sections of DNA in vitro. Restriction enzymes like EcoRI that generate sticky ends of DNA are often used to cut DNA prior to ligation, as the sticky ends make the ligation reaction more efficient. 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.
- Pingoud, A., Jeltsch, A. (2001). "Structure and function of type II restriction endonucleases". Nucl. Acids Res. 29 (18): 3705–3727. doi:10.1093/nar/29.18.3705. PMC 55916. PMID 11557805.
- Kurpiewski, M. R., Engler, L. E., Wozniak, L. A., Kobylanska, A., Koziolkiewicz, M., Stec, W. J, Jen-Jacobson, L (2004). "Mechanisms of coupling between DNA recognition and catalysis in EcoRI endonucleases". Structure 12: 1775–1788. doi:10.1016/j.str.2004.07.016. PMID 15458627.
- Bitinaite, J., D. A. Wah, Aggarwal, A. K., Schildkraut, I. (1998). "FokI dimerization is required for DNA cleavage". Proc Natl Acad Sci U S A 95 (18): 10570–5. doi:10.1073/pnas.95.18.10570. PMC 27935. PMID 9724744.
- Kim, Y. C., Grable, J. C., Love, R., Greene, P. J., Rosenberg, J. M. (1990). "Refinement of EcoRI endonuclease crystal structure: a revised protein chain tracing". Science 249 (4974): 1307–1309. doi:10.1126/science.2399465. PMID 2399465.
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