Sandbox 666: Difference between revisions

EcoRI is composed of two homodimers, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) 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 <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet, which is surrounded  by <scene name='Sandbox_666/Helix/1'>two α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.

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<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.

Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. 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<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.

Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. 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 <scene name='Sandbox_666/Grey_protein/3'>here</scene> .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/EXK motif. For EcoRI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD⁹¹ …E¹¹¹AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).