E. coli 6S RNA complexed to RNA polymerase maintains product RNA synthesis at low cellular ATP levels by initiation with noncanonical initiator nucleotides

6S RNA release is enabled in vitro by NCINs when ATP is absent. Native shift assay of internally radiolabeled synthetic 6S RNA^M releasing from RNAP, relative to free 6S RNA^M and 6S RNA^M:RNAP:σ⁷⁰ controls (”Free” and “Bound”). IN refers to each “initiator nucleotide”: ATP, NAD⁺, NADH, and FAD. Two negative controls lacking ATP and NCIN were prepared: “free” (6S RNA^M) and “bound” (6S RNA^M:RNAP:σ⁷⁰ complex). ATP was then added at 150 µM and 1000 µM as two positive controls. Each NCIN reaction was performed in the absence of ATP (“−ATP“) for 32 min using the indicated concentrations of NCIN. Samples were normalized by 6S RNA^M specific activity and resolved in a 5% native gel containing 5% glycerol at 4°C.