Significant conformational flexibility throughout the bridge helix is indicated by the presence of helix-destabilizing glycine residues at three to four conserved locations (Figure 1b). To detect regions in which the helix may be transiently disrupted during the nucleotide-addition cycle, Weinzierl [7] systematically substituted proline at every position in the helix. Most proline substitutions dramatically decreased total RNA synthesis on nicked calf-thymus DNA (the assay used in the robotic method). However, two substitutions, at positions 808 and 824 directly adjacent to conserved glycine residues (Cα spheres in Figure 1b), had the opposite effect of actually increasing total RNA synthesis. Interestingly, these positions correspond to the locations of naturally occurring prolines in the bridge helices of some bacterial RNA polymerases (for example, from Bacillus subtilis) or the newly described plant RNA polymerases IV and V (for example, from Arabidopsis thaliana). Thus, kinking focused at these two points of the bridge helix (Cα spheres in Figure 1b) appears not just to be tolerated, but to be stimulatory for RNA synthesis when facilitated by the presence of a proline residue.

Extension of these findings to investigate the curious presence of a deletion of two amino acids in the bridge helix of plant RNA polymerase IV (corresponding to the looped-out region proposed to act as a translocation pawl and shown as Cα-Cβ sticks in Figure 1b) led the author to a remarkable discovery that calls into question the translocation-pawl model. He reasoned that this deletion would radically twist the helix backbone and disrupt any coordinated looping-unlooping oscillations in the shortened region. Interestingly, the archaeal bridge helix tolerated a similar deletion without significant loss of activity not only at the polymerase IV location but also throughout the central portion of the bridge helix (white in Figure 1b). Lesser, but still significant, activity was observed in deletions near the amino-terminal proline substitution (gray in Figure 1b), whereas complete loss of activity was observed in deletions just amino- or carboxy-terminal to the proline substitutions (blue in Figure 1b). Bridge-helix regions that tolerated two-amino-acid deletions also tolerated proline substitution with only partial loss of activity. These results led Weinzierl to conclude that the proposed pawl-like function of the bridge helix or other proposed roles of this segment of the helix, such as contacting the template base, require re-evaluation because they are either redundant or do not exist for the archaeal helix.

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 (H_N and H_C), on the basis of the effects of the proline substitutions that increase polymerase activity (Cα spheres in Figures 1b and 2). H_N and H_C 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 H_N facilitates catalysis.

The H_N-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 H_C-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 H_N and H_C. 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 H_N and H_C 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 H_N and H_C. 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-H_N interaction [8] and with the general tolerance of the region between H_N and H_C 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.

The results of Weinzierl's tour de force mutagenesis of the bridge helix [7] yield several important ideas about its function. Careful testing of predictions based on these ideas is now necessary to advance understanding of RNA polymerase structure and function. These predictions include: that significant conformational changes can occur in the amino-terminal portion of the bridge helix; that bridge-helix movements mediate changes in RNA polymerase activity via modules that interact with the amino- and carboxy-terminal portions of the bridge helix; and that the bridge-helix looping originally observed in DNA-free RNA polymerase structures plays no vital role in translocation. These tests will require examination of the bridge-helix variants described by Weinzierl [7] using biochemical assays that detect individual steps in the nucleotide-addition cycle, or of homologous alterations in other RNA polymerases for which a wider range of in vitro assays specific for individual steps in the cycle is available. The nonspecific RNA-synthesis assay used in the robotic approach does not identify which step in the cycle is stimulated by H_N and H_C proline substitutions or inhibited by other alterations; even template engagement could be affected, as faster recycling of RNA polymerase could also increase RNA yield. Thus, much important biochemistry remains before we will fully understand RNA polymerases. Among the most important objectives should be to devise an assay that unambiguously and directly reports effects on translocation. A second key goal should be the determination of additional crystal structures of DNA-bound RNA polymerases that capture more extensive conformational changes, such as clamp opening, that might reveal the predicted changes in bridge-helix conformation.