Structural Biochemistry/Enzyme Catalytic Mechanism/EcoRI

Overview
Escherichia coli (EcoRI) is an endonuclease enzyme that is located in the restriction modification system. EcoRI is also isolated from the E. coli strain. EcoRI DNA is responsible for the specificity of E. coli strains harboring the fi+ drug resistance transfer factor RTFI. This is due to the introducing of two single strands staggered within the recognition sequence. Since EcoRI enzymes are considered to be the simplest sequence-specific DNA enzymes, they are well suited for the study of DNA sequence recognition by proteins. It is also possible to potentially compare two proteins interactions with the same DNA sequence using this enzyme.

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It is extracted from strains of E. coli and is part of the restriction modification system. In molecular biology, it is used as a restriction enzyme and it creates sticky ends with 5' end overhangs. The nucleic acid sequence where the enzyme cuts is GAATTC and the complementary sequence is CTTAAG. EcoRI is 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 cause 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. A procedure for large scale isolation of Escherichia coli RI endonuclease in high yield has been developed. The purified enzyme is homogeneous as illustrated by polyacrylamide gel electrophoresis and analytical sedimentation.