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

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.

Identification and Structural Modeling of the RNA Polymerase Omega Subunits in Chlamydiae and Other Obligate Intracellular Bacteria

“…The AlphaFold-Multimer-predicted three-dimensional structures of complexes of CTL0286 with the C. trachomatis RNAP β′ subunit and of CTL0286 with the C. trachomatis RNAP β′ and β subunits exhibit strong similarity to the experimental three-dimensional structures of ω-β′ and β′-β complexes ( Fig. 4 and 5 ) ( 86 , 87 ). The identification of CTL0286 as the C. trachomatis ω subunit demonstrates the power of the use of combinations of sequence similarity analysis, synteny analysis, and AlphaFold and AlphaFold-Multimer analyses for identifying proteins “missing” from proteomes and annotating functions of hypothetical proteins in proteomes.…”

“…In the resulting predicted structure of CTL0286 ( Fig. 3A ), the N-terminal region (residues 1 to 58) contains three α helices (α1 [residues 9 to 15], α2 [residues 19 to 36], and α3 [residues 44 to 55]) that correspond to three α helices present in structurally characterized ω subunits ( 26 , 86 – 96 ), and the C-terminal region (residues 58 to 100) is disordered. Three-dimensional-structure similarity searches of the AlphaFold prediction for full-length CTL0286, performed on the DALI server ( 97 , 98 ), identified bacterial ω subunits as the three top hits ( Fig.…”

Identification and structural modeling of the chlamydial RNA polymerase omega subunit

“…AlphaFold-Multimer predicted three-dimensional structures of complexes of CTL0286, with C. trachomatis RNAP β' subunit, and of CTL0286 with C. trachomatis RNAP β' and β subunits, exhibit strong similarity to experimental three-dimensional structures of ω-β' and β'-β complexes [(Fig. 4, 5); (97,98)]. The identification of CTL0286 as the C. trachomatis ω demonstrates the power of use of combinations of sequence-similarity analysis, synteny analysis, and AlphaFold and AlphaFold-Multimer analysis for identifying proteins "missing" from proteomes and for annotating functions of hypothetical proteins in proteomes.…”

“…We first used AlphaFold to predict three-dimensional structure of CTL0286. In the resulting predicted structure for CTL0286, the N-terminal region (residues 1-58) contains three α helices (α1, residues 9-15 residues; α2, residues 19-36; and α3, residues 44-55) that correspond to three α-helices present in all structurally characterized ω subunits (26, 97, 98), and the C-terminal region (residues 58-100) are mostly disordered, similar to in structurally characterized ω subunits having lengths greater ∼60 amino acids (26, 97, 98). Three-dimensional-structure similarity searches of the AlphaFold prediction for full-length CTL0286, performed on the DALI server (90, 91), identified bacterial ω subunits as the three top hits, with Z-scores of 3.8, 3.4, and 3.1, for RNAP ω subunits of Clostridium difficile (99), Mycobacterium tuberculosis (100), and Bacillus subtilis (101), respectively (Table 2).…”

Recycling of Bacterial RNA Polymerase by the Swi2/Snf2 ATPase RapA

“…In bulk transcription reactions with purified RNAP and σ 70 , the addition of RapA strongly stimulates multi-round transcription cycling and transcript production, raising the possibility that it acts following termination (17,(22)(23)(24)(25). However, multiple other activities for RapA have been proposed, including stabilizing open-promoter complexes during transcription initiation (26), promoting nascent transcript release from elongation complexes (24), preventing binding of RNAP on non-promoter DNA (27), and removing stalled elongation complexes via backtracking (28).…”