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

Abolishing the 6S RNA hairpin increases pRNA length and release time. (A) The 6S RNA swapped construct (6S_S) conserves pairing interactions in the downstream region but lacks the ability to form a hairpin. The top strand in the 6S RNA (green box) was swapped with the bottom strand (blue box) to generate the 6S_S construct. (B) Radiolabeled 6S_S (6S_S*) release assay showing a dramatic accumulation of the S3 state. (Lanes 1–6) 6S_S RNA. (Lanes 7–12) 6S RNA. Time points are 2, 5, 10, and 30 min; F indicates free RNA; B, bound RNA. (C) pRNA lengths in the S3 and S4 states of 6S_S relative to 6S RNA S4 state. All pRNAs were initiated with γ−[³²P]-ATP. H indicates alkaline hydrolysis ladder. Samples were analyzed using 23% denaturing PAGE.