Structural basis of RNA polymerase recycling by the Swi2/Snf2 family of ATPase RapA in Escherichia coli

M Zuhaib Qayyum,Vadim Molodtsov,Andrew Renda,Katsuhiko S Murakami

doi:10.1016/j.jbc.2021.101404

M Zuhaib Qayyum, Vadim Molodtsov + Show 2 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101404

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Abstract

After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined post-termination complex (PTC), limiting the free RNAP pool and subsequently leading to inefficient transcription. In Escherichia coli, a Swi2/Snf2 family of ATPase called RapA is known to be involved in countering such inefficiency through RNAP recycling; however, the precise mechanism of this recycling is unclear. To better understand its mechanism, here we determined the structures of two sets of E. coli RapA–RNAP complexes, along with the RNAP core enzyme and the elongation complex, using cryo-EM. These structures revealed the large conformational changes of RNAP and RapA upon their association that has been implicated in the hindrance of PTC formation. Our results along with DNA-binding assays reveal that although RapA binds RNAP away from the DNA-binding main channel, its binding can allosterically close the RNAP clamp, thereby preventing its nonspecific DNA binding and PTC formation. Taken together, we propose that RapA acts as a guardian of RNAP by which RapA prevents nonspecific DNA binding of RNAP without affecting the binding of promoter DNA recognition σ factor, thereby enhancing RNAP recycling.

Highlights

Thomas Research Center, Department of Structural Biology, St Jude Children’s Research Hospital, Memphis, TN 38105, USA
The clamp primarily remains in its “open” conformation in the RNA polymerase (RNAP) core enzyme, but it closes upon formation of holoenzyme, open complex, and elongation complex (EC) with DNA/RNA accommodated in the main channel of RNAP
Based on the structural findings and results of ATPase assay of RapA and DNA-binding assay of RNAP, we propose that RapA functions in RNAP recycling by acting as a guardian; RapA prevents nonspecific association of RNAP with DNA to maintain pool of free RNAP

Summary

RESEARCH ARTICLE

To better understand its mechanism, here we determined the structures of two sets of E. coli RapA– RNAP complexes, along with the RNAP core enzyme and the elongation complex, using cryo-EM These structures revealed the large conformational changes of RNAP and RapA upon their association that has been implicated in the hindrance of PTC formation. The clamp primarily remains in its “open” conformation in the RNAP core enzyme, but it closes upon formation of holoenzyme, open complex, and EC with DNA/RNA accommodated in the main channel of RNAP. These studies proposed a mechanism of RNAP recycling wherein HelD inserts two domains deep into the main and secondary channels of RNAP and promotes the clamp opening, destabilizing the EC and releasing DNA and RNA from RNAP. Based on the structural findings and results of ATPase assay of RapA and DNA-binding assay of RNAP, we propose that RapA functions in RNAP recycling by acting as a guardian; RapA prevents nonspecific association of RNAP with DNA to maintain pool of free RNAP

Results and discussion

RapA constraints nonspecific association of RNAP with DNA

Protein purifications

Core RNAP

Model building and refinement

ATPase assay