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

NAD(H) and FAD-modified pRNA products produced in vitro. (A) Denaturing gel analysis of NAD(H) containing pRNAs produced in vitro. (B) APB denaturing gel analysis of the same samples. Both gels in panels A and B were resolved using 20% denaturing PAGE. (C) RNA processing by CIP, RppH and NudC. The shared adenosine diphosphate is shown in black, whereas the differing upstream moiety is shown in red. X refers to a 5′ cap. (D) 5′ processing of in vitro synthesized pRNA using γ-³²P ATP and processed with CIP and RppH. (E) 5′ processing of in vitro synthesized α-³²P UTP radiolabeled pRNA species using CIP, RppH, and NudC. “(pp)A” refers to the 5′ terminal end of pRNAs containing either “ppA” or “A” (5′-OH), as these pRNA species migrate with similar velocities. 5′-triphosphorylated (“pppA”) pRNAs (ATP ± CIP conditions) shown in panel E run faster than A-pRNAs, given that pppA–pRNAs have more negative charges. The pA–pRNAs migrate faster than the pppA–pRNAs (panel E, lanes ATP ± RppH) due to less weight. These band patterns reflect a mixture of pRNAs initiated with a capped adenosine (slower mobility; NAD[H] or FAD) or ATP (fast mobility). Low amounts of HNppA–pRNA (NADH–pRNA) band are likely due to 5′ oxidation, resulting in ⁺NppA–pRNA (NAD⁺-pRNA). The ppA–pRNAs are assumed to have come from cap degradation. Samples in panels D and E were normalized to the same pRNA specific activity after Mango purification and resolved using 23% denaturing PAGE containing 0.25% APB.