Sandbox 666

From Proteopedia

Revision as of 13:53, 29 December 2011

Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme Eco RI.

Presentation

EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium, which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of EcoRI recognition sequences.

Reaction

EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor, which is the divalent ion Mg²⁺. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.

Structure

EcoRI is composed of two homodimers, so it has two identical subunits (Representation of one ) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core, which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five strands sheet, which is surrounded by ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.^[1]

In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and a new interpretation that alters the assignment of specific amino acid residues to some of the original features^[2]. In the new model,the Inner arm is the extended chain motif (Met¹³⁷to Ala¹⁴²)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone^[3]. The outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).

The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg²⁰⁰, Glu¹⁴⁴ and Arg¹⁴⁵. These aminoacids are shown in red .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively.

The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…(D/E)XK motif. For EcoRI, is PD⁹¹ …E¹¹¹AK and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).

The catalytic core of Eco RI with the target DNA

There are several non-contiguous structural elements, which are involved in DNA recognition:

   Four helix (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones
   An extended peptide chain running through the major groove forms the specific contacts of the enzymes to the DNA.
   One ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts)
   Two arms, which establish contacts with DNA backbones outside the recognition sequences.

This binding by the major groove is due to the position of scissile phosphodiester bonds.

Application of EcoRI in molecular biology

Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.

Links

[1] 1ERI in the Protein Database (PDB)

References

1. ↑ Refinement of EcoRI endonuclease crystal structure: a revised protein chain tracing. Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.
2. ↑ Refinement of EcoRI endonuclease crystal structure: a revised protein chain tracing. Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.
3. ↑ Refinement of EcoRI endonuclease crystal structure: a revised protein chain tracing. Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.
4. ↑ Structure and function of type II restriction endonucleases Alfred Pingoud, Albert Jeltsch Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727. PMCID: PMC55916

Contributor

Raphaël BILGER Virginie GROSBOILLOT