DNA Polymerase I: Difference between revisions
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In the structure on the left, the crystal had been soaked in a solution of dideoxy-CTP (ddCTP), which the enzyme had added to the 3' end of the primer chain (shown in space-filling form with C green), where it forms a base pair with the a template G. This terminates further primer extension due to the absence of a 3'-OH group at the 3' end of the primer strand. Nevertheless, a ddCTP (shown in space-filling form with C yellow) binds to the enzyme active site at the 3' end of the primer in a base pair with a template G as if it were preparing to add to the 3' end of the primer. In the structure on the right (2ktq), the ddCTP in the enzyme's active site had been depleted by soaking the crystal in a ddCTP-frree solution. Comparison of these two structures reveals that the structure on the left, the so-called closed conformation, differs from the that on the right, the so-called open conformation, by a hinge-like motion of the fingers domain away from the polymerase active site. The rest of the protein remains very nearly unchanged. This is more readily seen in the morph between the closed and open structures (in which, for technical reasons, the ddCTP in the closed conformation is not shown). | In the structure on the left, the crystal had been soaked in a solution of dideoxy-CTP (ddCTP), which the enzyme had added to the 3' end of the primer chain (shown in space-filling form with C green), where it forms a base pair with the a template G. This terminates further primer extension due to the absence of a 3'-OH group at the 3' end of the primer strand. Nevertheless, a ddCTP (shown in space-filling form with C yellow) binds to the enzyme active site at the 3' end of the primer in a base pair with a template G as if it were preparing to add to the 3' end of the primer. In the structure on the right (2ktq), the ddCTP in the enzyme's active site had been depleted by soaking the crystal in a ddCTP-frree solution. Comparison of these two structures reveals that the structure on the left, the so-called closed conformation, differs from the that on the right, the so-called open conformation, by a hinge-like motion of the fingers domain away from the polymerase active site. The rest of the protein remains very nearly unchanged. This is more readily seen in the morph between the closed and open structures (in which, for technical reasons, the ddCTP in the closed conformation is not shown). | ||
This, together with other experimental measurements, indicates that Klentaq1 rapidly samples the available dNTPs in its open conformation, but only when it binds the correct dNTP in a Watson–Crick pairing with the template base does it form the catalytically competent closed conformation. In addition, note how the template G that base pairs with the ddCTP in the closed conformation, moves away from the active site in the open conformation, in which it has no base pairing partner. | This, together with other experimental measurements, indicates that Klentaq1 rapidly samples the available dNTPs in its open conformation, but only when it binds the correct dNTP in a Watson–Crick pairing with the template base does it form the catalytically competent closed conformation. In addition, note how the template G that base pairs with the ddCTP in the closed conformation, moves away from the active site in the open conformation, in which it has no base pairing partner. | ||
<scene name='Sandbox_dvoet/DNA_polymerase/Klentaq1-closed_closeup/3' target='Closed' >closed | A closeup of the active site region in the <scene name='Sandbox_dvoet/DNA_polymerase/Klentaq1-open_closeup/6' target='Open' >open conformation</scene> (''right'') reveals that the side chain of the conserved Tyr 671 (colored with C pink) is stacked on top of the template G that forms a base pair with the bound ddCTP, where it apparently participates in verifying that a Watson–Crick base pair has formed. In the <scene name='Sandbox_dvoet/DNA_polymerase/Klentaq1-closed_closeup/3' target='Closed' >closed conformation</scene> (''left''), Tyr 671, which is part of the fingers domain, has moved aside, presumably to permit the active site to form about the incoming dNTP (satisfy yourself that the Tyr 671 side chain is stacked on the template G in the open form but not in the closed form). | ||
<scene name='Sandbox_dvoet/DNA_polymerase/ | <scene name='Sandbox_dvoet/DNA_polymerase/Morphtest1/1'>morph test scene</scene> | ||
==References== | ==References== | ||
<references/> | <references/> | ||