DNA Polymerase I: Difference between revisions
Donald Voet (talk | contribs) |
Donald Voet (talk | contribs) |
||
| Line 61: | Line 61: | ||
Here, Klentaq1's N-terminal, palm, fingers and thumb domains are yellow, magenta, green, and blue, respectively. The DNA is drawn in stick form colored according to atom type (template C cyan, primer C green, N blue, O red, and P orange). | Here, Klentaq1's N-terminal, palm, fingers and thumb domains are yellow, magenta, green, and blue, respectively. The DNA is drawn in stick form colored according to atom type (template C cyan, primer C green, N blue, O red, and P orange). | ||
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, 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 <scene name='DNA_Polymerase_I/Morphtest/3'>morph between the closed and open structures</scene> ('left'', 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, 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 <scene name='DNA_Polymerase_I/Morphtest/3'>morph between the closed and open structures</scene> (''left'', 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. | ||