EcoRI- Definition, Structure, Uses and FAQs for NEET Exam

EcoRI (which is pronounced as "eco R one") is a restriction endonuclease enzyme that is found in E. coli bacteria. It's a restriction enzyme that cuts/cleaves DNA double helices into pieces at particular sites as a result of the restriction-modification mechanism. Protective methylation at the specified target site nucleotides, which is put in place immediately after replication, ensures that the bacteria's DNA remains untouched.

EcoRI Full Form: The Eco portion of the enzyme's name comes from the species where it was isolated - "E" stands for "Escherichia" and "co" stands for "coli" - while the R stands for the specific strain, in this case, RY13, and the I stands for "first-ever enzyme extracted from this strain."

Eco-RI is a type II restriction enzyme that binds to double-stranded DNA and cleaves it at particular base pairs. It accomplishes this cleavage by accelerating the hydrolysis of the phosphodiester links in the target DNA. These enzymes' specificity is usually limited to four to six base pairs, and they require magnesium cations as cofactors for catalysis.

It has been used as a restriction enzyme in molecular biology. With the 5' end overhangs of AATT, EcoRI generates four nucleotide sticky ends. The enzyme breaks the nucleic acid recognition sequence (EcoRI recognition sequence) G↓AATTC, which does have a palindromic, complementary sequence of CTTAA↓G. Certain restriction enzymes may leave 3' overhangs or blunt ends without any overhangs, based on their break sites (EcoRI recognition sequence). EcoRI is a 377-amino-acid protein with a molecular weight of 31 kDa. It's a homodimer, to be precise.

Types of Restriction Endonucleases

Natural restriction endonucleases are classified into four classes based on their composition and enzyme cofactor needs, as well as the nature of their target sequence and the position of their DNA cleavage site relative to the target sequence (Types I, II, III, and IV). Restriction enzyme DNA sequence analyses, on the other hand, reveal significant variation, implying that more than four types exist.

All enzymes recognize certain short DNA sequences and cleave DNA into specific pieces with 5'-phosphates at the ends. As listed below, their recognition sequence, subunit composition, cleavage site, and cofactor needs differ:

• Type I enzymes cleave outside of the recognition site; they require ATP and S-adenosyl-L-methionine to function; multifunctional protein with restriction digesting and methylase activity.

• Type II enzymes cleave inside or within a short-defined distance from a recognition site; majority require magnesium; single function (restriction digestion) enzymes do not require methylase.

• Type III enzymes cut at small distances from a recognition site; they require ATP (but not hydrolyze it); S-adenosyl-L-methionine stimulates but is not required; they form a complex with a modification methylase.

• Modified DNA, such as methylated, hydroxymethylated, and glucosyl-hydroxymethylated DNA, is targeted by Type IV enzymes.

• Guide RNAs are used by Type V enzymes (gRNAs)

Ecori Thermo

• The EcoRI restriction enzyme from Thermo Scientific recognizes GAATTC sites and slices well at 37°C with its buffer. 

• Conventional restriction endonucleases from Ecori Thermo Scientific are a wide set of high-quality restriction enzymes that have been designed to function in one of the Five Buffer System's buffers. In contrast, the universal Tango buffer is included for dual digestion ease. 

• In the prescribed buffer and chemical reactions, many of the enzymes show 100% activity. 

• Thermo Scientific restriction enzyme reaction buffers produce mixtures of BSA, which improves the stabilization of several enzymes and links contaminants in DNA preparations, ensuring consistent results.

Structure of EcoRI

1. Primary Structure: EcoRI, like several other restriction endonucleases, does have the PD..D/EXK motif within its active site. It has a molecular weight of 30928.1 Daltons and is made up of 277 amino acids.

2. Secondary Structure: The globular protein is 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. Antiparallel and parallel motifs are found in the beta-sheets, which help in strand scission and sequence identification in the helices.

3. Tertiary Structure: It only has one alpha subunit that binds to one DNA strand. The polar and nonpolar residues are organized on the inside and outside, with nonpolar amino acids on the inside and polar amino acids on the outside.

4. Quaternary Structure: It's 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.

The enzyme can be regarded as a homodimer made up of one globular domain of the alpha/beta architecture and weighs around 31 kilodalton subunit. When bound, each subunit has a loop that protrudes from the globular domain and coils across the DNA. A green line indicates the cutting pattern at the EcoRI recognition site.

EcoRI has indeed been co-crystallized with the series that it cuts normally. The configuration of the complex 1QPS was solved using this crystal. The resolved crystal structure reveals that the enzyme homodimer's subunits symmetrically communicate with DNA. Two -helices within each subunit merge to create a four-helix package in the complex

Restriction Endonucleases

A restriction enzyme, also known as a restriction endonuclease or restricts, is an enzyme that cuts/ cleaves DNA into pieces at or close to certain restriction sites throughout molecules. Restriction enzymes are a subset of the larger endonuclease enzyme family. 

Restriction enzymes are divided into five groups based on their structure and whether they cut or do not cut their DNA substrate just at the recognition site or if the recognition and cleavage centers are distinct. Both restriction enzymes cut DNA twice, usually through every other sugar-phosphate backbone (i.e. each strand) of the double helix.

These enzymes are present in bacteria as well as archaea, and they operate like a virus defense mechanism. Restriction enzymes within a prokaryote exclusively break up foreign DNA inside a process known as restriction digestion, while host DNA is covered by a modification enzyme that alters the prokaryotic DNA and prevents cleavage. The restriction alteration mechanism is made up of these two processes.

Artificial Restriction Enzyme

By connecting a natural or designed DNA-binding domain to a nuclease domain, artificial restriction enzymes can be generated (often the cleavage domain of the type IIS restriction enzyme FokI). These synthetic restriction enzymes can target vast DNA locations and bind to specific DNA sequences (up to 36 bp). Zinc finger nucleases are the most widely utilized artificial restriction enzymes. They are most typically used in genetic engineering applications, but they can also be used in traditional gene cloning applications. The DNA binding domain of TAL effectors is the basis for other artificial restriction enzymes.

Uses

• Restriction enzymes like EcoRI have been used in a wide range of molecular genetics techniques such as DNA screening, cloning, and in vitro deletion of parts of DNA.

• Restriction enzymes that produce sticky ends of DNA, such as EcoRI, are frequently used to slice DNA before ligation because the sticky ends speed up the ligation reaction.

• Subject to the reaction conditions, EcoRI may show non-site-specific cutting, also recognized as star activity. 

• Lower sodium concentrations, higher glycerol concentrations, unnecessary quantities of enzymes involved in the reaction, elevated pH, and contamination with some organic solvents are all conditions that can trigger star activity while using EcoRI.