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

Anti-σ⁷⁰ antibody supershift assay showing S3 state is devoid of σ⁷⁰. (A) 25 nM radiolabeled 6S RNA bound to 200 nM holoenzyme was incubated with mouse anti-σ⁷⁰ antibody and was released with 147 μM of each NTP and 3.75 mM MgCl₂. F indicates free 6S; B, bound Eσ⁷⁰ complex; B′, Eσ⁷⁰ Ab; and SS, super shifted band. Time points of 2, 4, 8, 16, and 32 min were loaded in a 5% native gel with 5% glycerol. (B) The 25 nM body-labeled 6S RNA bound to 200 nM holoenzyme was incubated with or without mouse anti-σ⁷⁰ antibody as indicated and was released with 147 μM of each CTP, GTP, and UTP; 1.5 mM MgCl₂; and 25 μM AU. Time points of 1, 2, 4, 8, 16, and 32 min were loaded as in panel A.