Together, Weinzierl's findings point instead to critical roles of bridge-helix segments that contact flexible loops in the polymerase on either side of the active site, the downstream DNA channel, and the secondary channel, through which NTPs enter the active site. He designates these segments as amino- and carboxy-terminal hinges (HN and HC), on the basis of the effects of the proline substitutions that increase polymerase activity (Cα spheres in Figures 1b and 2). HN and HC are adjacent both to highly conserved glycines that are likely to facilitate bridge-helix distortions and to regions that do not tolerate alteration (blue in Figures 1b and 2). Like Pro-Gly sequences that occur at the hinge points of the trigger loop-trigger helix transition, these hinge regions may facilitate helix distortions important to RNA polymerase function. Recently, Seibold et al. [8] also proposed that helix bending at HN facilitates catalysis.
The HN-proximal bridge-helix segment contacts four conserved loops in the polymerase that form a cap to the helix and that, in turn, make critical contacts to: the trigger helices (β'/RPB1 F-loop; light pink in Figure 2) [9]; the downstream fork junction of duplex and melted DNA (β/RPB2 fork loop; light blue in Figure 2) [3, 8]; the NTP substrate (β/RPB2 D-loop; blue in Figure 2) [3]; and the nascent RNA, especially backtracked RNA (a β/RPB2 helix and loop termed the 'link domain' by Weinzierl [7]; green in Figure 2) [3, 10]. The HC-proximal bridge-helix segment contacts the clamp domain and switch regions 1 and 5 (red and purple in Figure 2) in an anchor that changes conformation when the clamp changes position or upon formation of the trigger helices (Figure 2). When the trigger helices form, contacts of the bridge helix to the cap are reduced, consistent with movement of the central portion of the helix toward the trigger helices by 1.5 Å (Figure 2) [3]. Although this movement is modest, larger movements of the bridge helix occur in a wedged intermediate generated by α-amanitin binding to RNA polymerase II [11]. Facilitating these bridge-helix movements by increasing flexibility may explain the superactivity of proline substitutions at HN and HC. Furthermore, it is likely that these regions undergo other, and quite possibly larger, changes during steps of the nucleotide-addition cycle, including translocation, that remain to be captured by crystal structures. Thus, kinking of the bridge helix at HN and HC may allow it to coordinate conformational coupling between the two sides of the polymerase cleft in ways that remain to be elucidated.
In this view, bridge-helix conformation influences formation of the trigger helices (and thus catalysis) in response to DNA and RNA sequence or transcription regulators that interact with the RNA polymerase clamp, cap, or anchor and affect bridge-helix conformation through HN and HC. Loops observed in the central portion of the helix may be a simple consequence of its inherent instability as a helix, which optimally poises it to modulate trigger-helix formation, rather than making loop-specific contacts (for example, as a ratchet pawl). Such a view is consistent with impairment of catalysis by substitutions that disrupt fork loop-HN interaction [8] and with the general tolerance of the region between HN and HC to significant alterations such as the two-amino-acid deletions [7] and helix-destabilizing substitutions [5], as the effects on mediating regulatory signals may not be evident in a nonspecific transcription assay. It would also explain how the divergent bridge-helix sequences found in the plant RNA polymerases IV and V could accommodate robust DNA-dependent RNA synthesis. It bears emphasizing, however, that roles of the bridge helix in controlling catalysis via effects on formation of the trigger helices and in facilitating translocation are not mutually exclusive.