EcoRI

EcoRI (pronounced “eco R one”) is just a restriction endonuclease enzyme found in the E. coli bacteria. It’s a restriction enzyme that functions to cleave DNA double helices into pieces at certain locations as part of the restriction modification system. The Eco component of the enzyme’s name comes from the species from which it was isolated – “E” stands for “Escherichia” and “co” stands for “coli” – while the R stands for the particular strain, in this case RY13, and the I stands for “first enzyme isolated from this strain.” It is employed as a restriction enzyme in molecular biology. With the 5′ end overhangs of AATT, EcoRI generates four nucleotide sticky ends. The enzyme cuts the nucleic acid recognition sequence GAATTC, which possess a palindromic, complementary sequence of CTTAAG. Other restriction enzymes can leave 3′ overhangs or blunt ends with no overhangs, depending on their cut sites.

Structure of EcoRI Enzymes

Eco-RI has a primary structure of 277 amino acids and a molecular weight of 30928.1 Daltons. Given that the average amino acid possess a molecular mass of 100 Daltons, the molecular weight is extremely close to, but somewhat more than, the expected value. This indicates that the majority of amino acids are average, with a small percentage being over average. 7.77 is the isoelectric point, or the pH at which the molecule has no net charge.

The globular protein Eco-RI has a secondary structure made up of alpha helices, beta sheets, and 3/10 helices. The alpha helices make up the majority of the protein’s outside, while the 3/10 helices make up the majority of its inner. The beta sheets include parallel and antiparallel motifs that contribute in strand scission and sequence recognition, which takes place in the helices. Eco-RI contains around 20% beta sheets, and more than 50% alpha helices.

Eco-RI has only one alpha subunit that binds to one strand of DNA in its tertiary structure. The polar and nonpolar residues are organised on the inside and outside, with nonpolar amino acids on the inside and polar amino acids on the outside. By allowing exterior polar molecules to interact with the cell’s water-based environment rather than water molecules losing entropy by building ordered structures around nonpolar foreign residues, the hydrophobic effect and van der Waals interactions are maximised. The 3/10 helices, which are made entirely of polar and basic  amino acids in the interior, are an exception to the protein’s largely nonpolar interior.  

Eco-quaternary RI’s structure is a homodimer made up of several motifs, four of which are noteworthy. The extended chain motif stretches from Met-137 to Ala-142 and runs parallel to the DNA backbone through the main groove. This region is a mutational hotspot that interacts with pyrimidine nucleotides primarily. The beta bridge is made up of an alpha helix and a pair of antiparallel beta strands that joins 1 and 2 to span the distance between globular units. This bridge is one of the parts of the structure that holds the DNA backbone. The position of Glu-111 is crucial since mutations here can diminish the enzyme’s cleavage activity without affecting DNA binding. Finally, a beta-loop-alpha motif connects 3 and 4 and 4 and 5 to produce arms that wrap the DNA and contact the backbone. This last motif has a broad topological significance in nucleic acid binding.

Source of EcoRI Enzymes

Escherichia coli RY13 is the source of Eco RI. Eco RI is a restriction enzyme that makes a particular cut in DNA. These can be used to separate our chosen gene from a large number of others. Only one recognition site on the DNA is cleaved by a restriction enzyme. The palindrome sequence can be found on these sites. A palindrome sequence is one in which both strands have the identical base sequence. At ‘GAATTC,’ the restriction enzyme EcoRI breaks the DNA. Only one restriction endonuclease recognises a certain nucleotide sequence of DNA, and none of the others. In bacteria, restriction endonuclease enzymes are discovered. By cutting the genetic material of bacteriophage, these enzymes prevent it from growing in bacteria. As a result, these enzymes are derived from bacteria.

The HindIII enzyme was the first of this type to be found. More than 900 enzymes have now been found and extracted from various bacterium strains. The names of the bacteria from which restriction enzymes were extracted are used to nominate them. Eco RI is derived from the Escherichia coli RY13 strain. The letters ‘E’ stand for the genus Escherichia, ‘co’ for the species coli, and ‘R’ for the bacteria strain. The letter ‘I’ indicates that this was the strain’s first isolated enzyme. These enzymes produce DNA sticky ends, which are exploited in recombinant DNA technology. Both the plasmid and the DNA fragment that needs to be cleaved are treated with the same restriction enzyme.

Uses of EcoRI

Restriction enzymes are employed in cloning, DNA screening, and deleting portions of DNA in vitro, among other molecular genetics techniques. Restriction enzymes that generate sticky ends of DNA, such as EcoRI, are frequently employed to cut DNA prior to ligation because sticky ends improve the efficiency of the ligation reaction. In the synthesis of recombinant DNA, this technique is used to link donor and vector DNA. Depending on the reaction conditions, EcoRI can display non-site-specific cutting, often known as star activity. Low salt concentrations, high glycerol concentrations, excessive levels of enzyme present in the reaction, high pH, and contamination with specific organic solvents are all conditions that can cause star activity when employing EcoRI.

Conclusion

Restriction endonucleases that recognise specific nucleotide sequences can be used to cut genes from total genomic DNA. For detailed study, individual genes can be acquired and copied in quantity. Cloning is a technique that uses bacterial plasmids and viruses (phage) as carriers for cloned genes. Foreign DNA is joined to plasmid or phage vectors by DNA ligases, which can then proliferate within bacterial cells to build gene libraries. Cloned genes operate as probes in nucleic acid hybridization, detecting the presence of their native counterparts in complicated mixes of DNA or RNA.