What is EcoRI?

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EcoRI Restriction Enzyme

EcoRI (which is pronounced as "eco R one") is a restriction endonuclease enzyme that is found in the E. coli bacteria. It's a restriction enzyme which cuts/cleaves DNA double helices into pieces at particular sites as a result of the restriction modification mechanism.

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."

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 Thermo- 

  • The EcoRI restriction enzyme from Thermo Scientific recognises GAATTC sites and slices well at 37°C with its own 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 BSA, which improves the stabilization of several enzymes and links contaminants in DNA preparations, ensuring consistent results.

Structure

Primary Structure: EcoRI, like several other restriction endonucleases, does have the PD..D/EXK motif within its active site.

Tertiary and Quaternary Structure: 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 communicate with DNA in a symmetrical manner. 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 restrictase, is an enzyme which 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 as a virus defence 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.


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 which 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.

FAQ (Frequently Asked Questions)

1. What is the Optimal Temperature For Ecori Digestion?

Ans. The optimal temperature for ecori digestion is 37oC. EcoRI is highly sensitive to heat activation.

2. What's the Number of Fragments Generated By the Ecori Restriction Enzyme?

Ans. The number of fragments generated by the ecori restriction enzyme is four fragments.

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