Figure 4.

Structural changes at the active site during a single nucleotide addition cycle (Yin and Steitz, 2004). This figure shows the O helix with its phosphate binding K631 and R627, a β turn–β motif bearing the metal binding catalytic D812 and D537, template nucleotides in blue, the RNA primer terminus in green, as well as the P and N sites in green and pink ovals. (A) The NTP (red) is bound to the N site in position to be inserted with its metal-bound triphosphate moiety crosslinking the O helix to the active site aspartic acid residues. Template nucleotide i+1 (light blue) forms a base pair with the correct incoming nucleotide. (B) The product of the phosphoryl transfer reaction shows a Mg ion (blue) bound to PP_i (red), which crosslinks D531 to R677, thereby maintaining RNAP in an identical conformation as in the substrate complex. The 3′ end of RNA remains in the N site in a pre-translocation state. (C) Release of Mg-PP_i results in the loss of the link between the O helix and D531, which promotes the rotation of the O helix and translocation of the 3′ end of the RNA to the P site. The RNAP conformational change also places Y639 into the N site and positions the i+2 template nucleotide into the flipped-out, pre-insertion position. (D) A modeled NTP pre-insertion complex with NTP bound to the post-translocated RNAP. Although the base binding site is blocked by the side chain of Y639, the triphosphate binding site on the O helix is accessible.