Abstract
The 6S RNA in Escherichia coli suppresses housekeeping transcription by binding to RNA polymerase holoenzyme (core polymerase + σ⁷⁰) under low nutrient conditions and rescues σ⁷⁰-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 γ-proteobacteria. Deliberately slowing the intrinsic 6S RNA release rate by nucleotide feeding experiments reveals that σ⁷⁰ ejection occurs abruptly once a pRNA length of 9 nucleotides (nt) is reached. After σ⁷⁰ 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 σ⁷⁰ ejection. Rather, the pRNA size required to trigger σ⁷⁰ release increases from 9 nt to 14 nt. That a precise pRNA length is required to trigger σ⁷⁰ 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 γ-proteobacteria: First, its formation strips single-stranded "-10" 6S RNA interactions away from σ⁷⁰. 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.