A range of 6S RNA processing events are associated with pRNA production in early outgrowth time points. (A) RNA transcription and ribonucleases process the 5′ termini of the 6S RNA (Kim 2004) prior to or during loading into the inhibitory complex, which are then released by pRNA synthesis. The RNAP core is shown in teal, and σ70 is shown in blue. (B) cpRNAs as a function of outgrowth time. Each outgrowth cpRNA sample purified from MIII ssrS bacteria was loaded either without or with alkaline hydrolysis treatment so as to measure the cDNA size. Dideoxy size ladders were generated by RT extension of synthetic DNA primer 5′-ATC GGC TCA GGG G-3′ adapted from the expected pRNA sequence. cpRNAs were labeled with α-32P dATP using RT. (C) Individual bands found in the 4 min time point shown in panel B were excised and subjected to complete alkaline hydrolysis. Bands are ordered by decreasing cpRNA length from left to right. The cpRNA bands indicative of 5′-precursor and matured 6S RNA species are marked by bold length numbers in the gap between the two gels. All samples purified by Mango and PAGE were normalized by cpRNA concentration and resolved using 10% denaturing PAGE. (D) Observed cpRNA sizes are related to 5′ mature 6S RNA as well as primary transcripts initiated at promoters P2 and P1.
