E. coli 6S RNA release from RNA polymerase requires σ⁷⁰ ejection by scrunching and is orchestrated by a conserved RNA hairpin

Abstract

The 6S RNA in Escherichia coli suppresses housekeeping transcription by binding to RNA polymerase holoenzyme (core polymerase + s 70 ) under low nutrient conditions and rescues s 70 -dependent transcription in high nutrient conditions by the synthesis of a short product RNA (pRNA) using itself as a template. Here we characterize a kinetic intermediate that arises during 6S RNA release. This state, consisting of 6S RNA and core polymerase, is related to the formation of a top-strand ''release'' hairpin that is conserved across the g-proteobacteria. Deliberately slowing the intrinsic 6S RNA release rate by nucleotide feeding experiments reveals that s 70 ejection occurs abruptly once a pRNA length of 9 nucleotides (nt) is reached. After s 70 ejection, an additional 4 nt of pRNA synthesis is required before the 6S:pRNA complex is finally released from core polymerase. Changing the E. coli 6S RNA sequence to preclude formation of the release hairpin dramatically slows the speed of 6S RNA release but, surprisingly, does not alter the abruptness of s 70 ejection. Rather, the pRNA size required to trigger s 70 release increases from 9 nt to 14 nt. That a precise pRNA length is required to trigger s 70 release either with or without a hairpin implicates an intrinsic ''scrunching''-type release mechanism. We speculate that the release hairpin serves two primary functions in the g-proteobacteria: First, its formation strips single-stranded ''À10'' 6S RNA interactions away from s 70 . Second, the formation of the hairpin accumulates RNA into a region of the polymerase complex previously associated with DNA scrunching, further destabilizing the 6S:pRNA:polymerase complex.

In vitro characterization of 6S RNA release-defective mutants uncovers features of pRNA-dependent release from RNA polymerase in E. coli

“…This data suggests that the region cannot be easily changed and is consistent with previous findings that indicate that the region has a complex effect on release rate (Shephard et al 2010). In contrast, the region from positions 135 to 141, which forms the upper part of the predicted release hairpin's left arm (Panchapakesan and Unrau 2012), had a significantly higher mutational frequency of FIGURE 2. Round 9 release-defective 6S RNA consensus compared with binding and release selection.…”

“…The types of mutants isolated suggest the existence of several discrete stages in the release process that are consistent with the proposed scrunching model of 6S RNA release (Panchapakesan and Unrau 2012). In the present paper, we focus on the R9-33 isolate due to its ability to remain bound to the Eσ 70 in conditions that normally induce very rapid (t 1/2 ∼ 30 sec) release of a truncated 6S RNA control construct called T1 (Fig.…”

“…1, state S4). Consistent with this model, swapping the downstream top and bottom strands to prevent the formation of the release hairpin slows the 6S RNA release rate, and RNA release is only achieved after the synthesis of a much longer pRNA in vitro (Panchapakesan and Unrau 2012). Interestingly, other bacteria like Bacillus subtilis seem to change their secondary structure in the bottom strand of the LOB during release (Panchapakesan and Unrau 2012;Steuten and Wagner 2012), implying that, just as in DNA-dependent scrunching, the mechanism of 6S RNA release is intrinsically flexible and presumably highly dependent on 6S RNA sequence.…”

“…A product RNA (pRNA) is shown in dark blue and triggers the formation of a release hairpin formed from the conserved "-10" region and the top strand of the downstream helix. Based on the scrunching-dependent release model for 6S RNA (Panchapakesan and Unrau 2012).…”

“…A published model suggests that the process of 6S RNA release is analogous to the process of DNA-dependent transcriptional initiation (Panchapakesan and Unrau 2012). During transcriptional initiation, DNA "scrunching" (Cheetham and Steitz 1999) steadily packs downstream DNA into the Eσ 70 complex via the ratcheting effect of NTP polymerization (Ederth et al 2002).…”