Figure 7. Mfd motions during its nucleotide hydrolysis cycle cause significant RNA polymerase (RNAP) conformational changes.

The completion of the Mfd loading pathway culminates in the formation of C1(ATP) (Supplementary file 5). Mfd then cycles through five distinct states in the order proposed here (also see Supplementary file 5, Figure 4—figure supplement 1A, and Figure 7—video 1). In looping through this cycle, internal conformational changes of Mfd are relatively small (involving primarily the nucleotide-dependent shifts of D5(TD1) and D6(TD2) with respect to each other; see Figure 3), but Mfd and the upstream duplex DNA as a whole wobble back and forth by about 30° on the upstream face of the RNAP. These motions cause significant RNAP conformational changes quantified in the radar plot in the middle. Using C1(ATP) as a reference structure, the RNAP clamp of C3(adp) is opened 17.3° (schematically illustrated in the cartoon inset). The βlobe-Si1 domain of C5(ATP) is rotated 14.3° as illustrated.

Figure 7—video 1. The Mfd nucleotide hydrolysis cycle.

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The video starts with two orthogonal views of C1 (color coded as in Figure 7, with the RNA polymerase (RNAP) βlobe-Si1 colored in teal, and the RNAP clamp colored in purple, and each domain highlighted with a transparent molecular surface). The video cycles through the five structures of the Mfd-nucleotide hydrolysis cycle (C1 → C2 → C3 → C4 → C5). The current structure is highlighted in red (lower left). At C2 → C3 the motions of Mfd open the RNAP clamp, and at C4 → C5 the motions of Mfd twist the βlobe-Si1. After two cycles, two more cycles are shown but Mfd and the nucleic acids are removed to further highlight the motions of RNAP.