An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options

Abstract

Transcriptional pausing underpins regulation of cellular RNA synthesis but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear if swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here we use cryo-EM analysis of ePECs with different RNA-DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the post-translocated state at certain RNA-DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation.

Structural and functional basis of the universal transcription factor NusG pro-pausing activity inMycobacterium tuberculosis

“…Distortions of the BH and possible effects on translocation and active-site configuration have been noted previously (Bar-Nahum et al, 2005;Gnatt et al, 2001), including in the swiveled, PH-stabilized hisPEC (Kang et al, 2018a). Our results and analysis clearly reveal why, as postulated (Kang et al, 2022;Zhu et al, 2022), the swiveled state is incompatible with catalysis, and how, in addition to other PECspecific DNA interactions (Guo et al, 2018;Saba et al, 2019), swiveling prolongs transcriptional pauses. 10/21/2022 Pro-versus anti-pausing NusGs switch conserved side-chain contacts from the GL to nt-…”

“…combined with other recent results (Kang et al, 2022;Zhu et al, 2022) lead us to make the following conjectures: i) All the ECs, with or without MtbNusG, are post-translocated, while all the PECs, with or without NusGs, are half-translocated. We conclude that sequence characteristics of the upstreamand downstream-fork junctions of the RNA-DNA hybrid induce the half-translocated state (Larson et al, 2014;Saba et al, 2019).…”

“…A fundamental mechanism of transcriptional pausing in bacteria that involves consensussequence inhibition of DNA translocation and rotation of a subset of RNAP modules has emerged from recent studies of Eco RNAP (Abdelkareem et al, 2019; Guo et al, 2018; Kang et al, 2022; Kang et al, 2018a; Kang et al, 2019; Landick, 2021; Qian et al, 2021; Saba et al, 2019; Zhu et al, 2022). Despite evidence that the consensus pause sequence is recognized by many RNAPs (Gajos et al, 2021; Larson et al, 2014), it has remained unclear if the fundamental mechanism is as widespread as the consensus sequence and how swiveling could contribute to pausing in bacteria lacking the SI3 TL insertion.…”

“…Although SI3-like domains are present in the TLs of many bacterial RNAPs (Lane and Darst, 2010a), they are absent in others including Bsu and MtbRNAPs that also respond to PHs (Figures 1 and S1) (Yakhnin and Babitzke, 2010;Yakhnin et al, 2008). Swiveling is also associated with non-PHdependent pausing (Abdelkareem et al, 2019;Guo et al, 2018;Kang et al, 2022;Zhu et al, 2022). These observations raise key questions: i) do bacterial RNAPs that lack SI3 still swivel or is swiveling an evolutionary adaptation to the presence of SI3-like domains that allows some 10/21/2022 bacterial clades to tune their response to pause signals?…”

“…In Eco , pause states are also associated with swiveling, an RNAP conformational change in which a set of RNAP structural modules (comprising ~1/3 of RNAP) rotate with respect to the rest of the RNAP. The swiveled RNAP may favor half-translocation and be refractory to nucleotide addition (Kang et al, 2022; Zhu et al, 2022). The ePEC can rearrange further into longer-lived PECs by backtracking or via interactions of nascent RNA structures (pause hairpins, PHs) or trans-acting regulators.…”