Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ

Abstract

Factor-dependent transcription termination mechanisms are poorly understood. We determined a series of cryo-electron microscopy structures portraying the hexameric ATPase ρ on path to terminating NusA/NusG-modified elongation complexes. An open ρ ring contacts NusA, NusG, and multiple regions of RNA polymerase, trapping and locally unwinding proximal upstream DNA. NusA wedges into the ρ ring, initially sequestering RNA. Upon deflection of distal upstream DNA over the RNA polymerase Zinc-binding domain, NusA rotates underneath one capping ρ subunit, which subsequently captures RNA. Following detachment of NusG and clamp opening, RNA polymerase loses its grip on the RNA:DNA hybrid and is inactivated. Our structural and functional analyses suggest that ρ and other termination factors across life may utilize analogous strategies to allosterically trap transcription complexes in a moribund state.

NusG, an Ancient Yet Rapidly Evolving Transcription Factor

“…However, the ring closure activity of NusG may not be the main mechanism by which NusG stimulates Rho-dependent termination. Consistent with biochemical data (Schmidt and Chamberlin, 1984;Epshtein et al, 2010) and genome-wide mapping (Mooney et al, 2009a) that support persistent Rho-RNAP interactions, a recent cryo-EM analysis of the E. coli TEC under attack by Rho reveals seven complexes thought to represent sequential steps in the termination pathway (Said et al, 2020). During the initial binding to the TEC, Rho makes numerous contacts to the RNAP subunits, NusA and NusG NGN (Figure 4), but captures the nascent RNA transcript only later in the pathway.…”

“…λN stabilizes the elongation-competent state of RNAP, inhibiting the nascent RNA hairpin formation and its stabilization by NusA, supports the anti-backtracking and anti-swiveling action of the NusG NGN domain. In the λN-TAC, neither NusG domain can make contacts to Rho observed in the binary Rho-NusG complex (Lawson et al, 2018) and Rho-TEC (Said et al, 2020) structures. Consequently, in the λN-TAC, NusG antipausing activity is augmented while its termination-promoting activity is abolished.…”

“…During the initial binding to the TEC, Rho makes numerous contacts to the RNAP subunits, NusA and NusG NGN (Figure 4), but captures the nascent RNA transcript only later in the pathway. Once engaged, Rho induces dramatic conformational changes in RNAP and Nus factors, which ultimately trap a moribund TEC in which the clamp is wide open and the RNA 3 end is dislodged from the RNAP active site (Said et al, 2020), a model initially proposed by Nudler and colleagues (Epshtein et al, 2010). In this structurally defined pathway, NusG NGN assists Rho loading onto the RNA and then dissociates to allow for Rho-mediated RNAP clamp opening, whereas NusG KOW is invisible.…”

“…By preventing backtracking, an activity welldocumented in the case of NusG and RfaH (Svetlov et al, 2007;Herbert et al, 2010), NusG-like proteins facilitate processive transcription and promote genome stability. Recent functional and structural data suggest a molecular mechanism of enhanced RNAP processivity, in which the NGN domain loops out the non-template DNA, bringing the upstream and downstream DNA duplexes closer together (Turtola and Belogurov, 2016;Kang et al, 2018;Nedialkov et al, 2018), and establishes contacts to the upstream DNA duplex (Krupp et al, 2019;Said et al, 2020). Together, these interactions alter the upstream DNA trajectory ( Figure 5) and stabilize the upstream edge of the transcription bubble, which must melt to allow backtracking, explaining how NusG and RfaH inhibit backtracking (Svetlov et al, 2007;Herbert et al, 2010).…”

Allosteric activation of SARS-CoV-2 RdRp by remdesivir triphosphate and other phosphorylated nucleotides

“…The SARS-CoV-2 RdRp subunit composition and dynamics resemble those of structurally unrelated bacterial RNA polymerases (RNAP). Bacterial enzymes are composed of 4–7 subunits and are elaborately controlled by regulatory nucleic acid signals and proteins that induce conformational changes in the transcription complex, as revealed by many recent cryoEM studies ( Guo et al, 2018 ; Kang et al, 2018 ; Said et al, 2021 ). Notably, most natural and synthetic products that inhibit bacterial RNAPs alter protein interfaces or trap transient intermediates rather than block nucleotide addition and bind to many different sites ( Chen et al, 2020a ).…”