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

Abstract

Transcriptional pausing underpins the 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 whether swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here, we use cryo-electron microscopy (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 posttranslocated 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.

RNA polymerase SI3 domain modulates global transcriptional pausing and pause-site fluctuations

“…ΔSI3* RNAP increased ce-pause strength (1.4-fold greater than wild type) (Figure 3C ), in contrast to its reduced hs-pausing. We conclude that ΔSI3* also mimics ΔSI3 in increasing ce-pausing possibly by increasing folded-TL stabilization of the pretranslocated ce-PEC ( 16 ).…”

“…SI3 is known to mediate effects of PHs via swiveling, so this effect of ΔSI3* is as expected. In contrast, the ce-pause appears to favor a pretranslocated rather than swiveled state ( 16 ). Thus, we next probed how ΔSI3* RNAP responds to a ce-pause signal.…”

“…significant pauses in Figure 5G ) generated a sequence logo comparable to that of the ΔSI3-enhanced pauses (compare Figure 6A , top two panels). We hypothesize that SI3 reduces the strength of strong elemental pauses at which TL folding stabilizes a pretranslocated paused state ( 16 ) and that these pauses are favored by sequences with optimal matches to the ce-pause sequence. Hence, SI3 deletion increases pausing at these sites.…”

“…Additionally, our validation of hs-pauses establishes unambiguously that SI3 modulates different classes of pauses oppositely. Pauses resembling the consensus elemental pause are inhibited by ΔSI3, presumably because SI3 destabilizes the folded-TL pretranslocated ePEC state whereas it inhibited TL folding in the hairpin-stabilized, swiveled PEC ( 16 ).…”

“…SI3 is proposed to be a key connector between RNAP swiveling and TL conformational change in a PECs; SI3–jaw rotates as part of the swivel module and thus stabilizes the unfolded TL ( 15 , 20 , 22 ). Conversely, SI3 may stabilize the folded TL in pretranslocated PECs, which form on strong ce-pause sequences ( 16 ). Deletion of SI3 or disruption of its connection with other swivel-module parts greatly reduces hs-pause strength in vitro ( 20 , 22 , 23 ).…”