NIH Research Festival
FARE Award Winner
Members of the evolutionary conserved Swi2/Snf2 (switch/sucrose non-fermentable) family depend on their ATPase activity to mobilize nucleic acid-protein complexes for gene expression. In bacteria, RapA is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein that mediates RNAP recycling during transcription. It is known that the ATPase activity of RapA is stimulated by its interaction with RNAP. However, how the RapA-RNAP interaction activates RNAP is not known. Previously, we determined the crystal structure of RapA. The structure revealed the dynamic nature of its N-terminal domain (Ntd), which prompted us to elucidate the solution structure and activity of both the full-length protein and its Ntd-truncated mutant (RapAdN). Here, we report the solution structures of RapA and RapAdN, either ligand-free or in complex with RNAP determined by small-angle X-ray scattering. The solution structures reveal a new conformation of RapA, define the binding site of RapA on RNAP, and show that the binding sites of RapA and sigma70 on RNAP largely overlap. We also report the ATPase activity of RapA and RapAdN, in the absence or presence of RNAP. We conclude that the ATPase activity of RapA is inhibited by its Ntd but stimulated by RNAP in an allosteric fashion and that the conformational changes of RapA and its interaction with RNAP are essential for RNAP recycling. These findings outline the functional cycle of RapA, which increases our understanding of the mechanism and regulation of Swi2/Snf2 proteins. The new structural information also leads to a hypothetical model of RapA in complex with RNAP immobilized during transcription.
Scientific Focus Area: Structural Biology
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