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

In vivo NCINs are used for pRNA initiation as cells enter stationary phase. Preparation and diagnosis of the 5′ end of Mango-purified, PAGE-purified outgrowth cpRNAs radiolabeled with α-³²P dATP. (A) Triphosphate cpRNA ligation procedure. (I) RppH treatment. (II) 5′ RNA adapter (purple) ligation. (III) Separation of adapter ligation products and unligated cpRNA by 8% denaturing PAGE. (B) Capped cpRNA ligation procedure. (I) CIP treatment. (II) NudC treatment. Both ligation (III) and PAGE (IV) steps are the same as in II and III of panel A. (C) Quantification of ligation percentage from panel A triphosphate cpRNA procedure. (D) Quantification of ligation percentage from panel B capped cpRNA procedure. (E) Capped cpRNA APB gel analysis (1536 min of outgrowth, only). (I) APB Purification using 10% denaturing PAGE containing 0.8% APB. (II) CIP treatment. (III) NudC treatment. (IV) 10% denaturing PAGE containing 0.8% APB to resolve the cpRNAs. The histograms in panels C and D are determined from a single experiment.