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

Mango III-dependent purification of the 6S RNA:pRNA complex and pRNA-cDNA chimera generation. (A) Schematic Mango purification protocol adapted from Panchapakesan et al. (2017) to include pRNA-cDNA chimera (cpRNA) synthesis. (I) Crude MIII ssrS RNA extracts were prepared from chilled, methanol fixed cell culture samples. 6S RNA^M is shown in black with conserved regions in pink, and the MIII tag in orange. Hybridized pRNA in a 6S RNA^M:pRNA complex is shown in blue. Extraneous cellular RNA is shown in brown. (II) 6S RNA^M:pRNA complexes were immobilized on streptavidin magnetic beads (“S”) derivatized with TO1-Biotin (shown in green and light gray) and washed. (III) Reverse transcription (RT) generates bead-immobilized pRNA–DNA (cpRNA) chimeras. The cDNA of cpRNA is shown in light blue. (IV) Formamide elution recovers cpRNAs for further downstream analysis. (B) Secondary structure of a 6S RNA^M:cpRNA complex bound to streptavidin beads. Radioactive cpRNA was generated by spiking the RT step with α-³²P dATP. “B” of TO1-Biotin refers to biotin bound to streptavidin, and “TO1” refers to thiazole orange. For fully matured 6S RNA, this cpRNA is 44-nt-long. Dashed lines at the 6S RNA^M 5′ end (cpRNA 3′ end) imply potentially longer 6S RNA and cDNA lengths resulting from immature 6S RNA.