(a) Electrophoretic analysis of RNA associated with FLAG-Rne-containing degradosomes. RNA extracted from degradosomes (lane C), purified ribosomes (lane R), or total lysates of E. coli (lane T) was separated on 5% denaturing polyacrylamide gels and stained with ethidium bromide. Positions of 23S, 16S, and 5S rRNAs and tRNAs are shown. (b) Electrophoretic analysis of RNA associated with immunoaffinity-purified native Rne complexes. RNA extracted from FLAG-Rne-containing degradosomes and total lysates of E. coli (lanes C and T, respectively) from purified native Rne complexes (lane A) were analyzed as described for a. Lane P shows a background RNA bound to protein A-Sepharose coupled with the preimmune serum antibodies. (c) Clones of cDNA derived from rRNA fragments associated with FLAG-Rne degradosome. Inserts of cDNA clones aligned with previously determined sequences of 16S and 23S rRNA. Arrowheads indicate the positions of oligonucleotide primers.

Northern blot analysis of rRNA associated with degradosomes. RNA extracted from FLAG-Rne-containing degradosomes (lane C), from ribosomes (lane R), or total E. coli lysate (lane T) was loaded onto 5% denaturing polyacrylamide gel and resolved by electrophoresis. RNA was transferred to a membrane and probed with uniformly labelled RNAs complementary to 16S (a), 23S (b), and 16S and 23S (c and e). (d) Results of Northern blot analysis where 9S antisense riboprobe was used. (e) Results of Northern blot analysis of RNA associated with immunoaffinity-purified native Rne complexes (lane A; see Fig. 1b). Positions of 23S, 16S, and 5S rRNAs are shown.

Detection of RNAI and RNAI₋₅ by primer extension analysis of degradosome RNA components. Degradosome expression was induced in BL21(DE3) strain, containing pRE196. Total RNA (lane t) and RNA components from degradosome (lane c) were analyzed. The positions of pppRNAI, RNAI₋₅, and RNAI decay intermediates are shown.

(a) Cleavage of total E. coli RNA by FLAG-Rne degradosomes. Total E. coli RNA was incubated in RNase E reaction buffer for 15 min at 30°C without (lane 1) or for 5 or 15 min with degradosomes (lanes 2 and 3). Samples for lanes 5–7 were prepared similarly, but reaction buffer was additionally supplemented with ATP and phosphate and incubation was done at 37°C. Lane 4 contains RNA components of degradosome. RNA was separated on gel and stained as described in Fig. 1a. Positions of 23S, 16S, and 5S rRNAs are shown. (b) Primer-extension analysis of degradosome RNase E cleavages. Total E. coli RNA was incubated in RNase E reaction buffer for 0 or 15 min without degradosomes (lanes 1 and 2) at 30°C. Incubations were performed for 15 min at the permissive temperature with degradosomes containing wild-type Rne (lane 3) or mutant (rne-3071) Rne (lane 4) or for 15 min at the nonpermissive temperature (44°C) with mutant Rne (lane 5). Lane 6 contains products from a primer-extension reaction of the RNA components isolated from FLAG-Rne degradosomes. Lanes 7–10 contain DNA sequencing ladder. Asterisks indicate fragments produced by RNase E. Aliquots of degradosome were incubated in RNase E buffer for 15 min at 30°C without addition of total RNA (lane 11). (c) Segments of folded 16S and 23S rRNAs (refs. 38 and 39) showing the identified cleavage sites.

Cleavage of RNA components by FLAG-Rne containing degradosomes. (a) RNA extracted from degradosome was incubated in RNase E reaction buffer supplemented with ATP and phosphate for 15 min at 37°C without (lane 2) and for 5 and 15 min with degradosome (lanes 3 and 4). E. coli tRNA was added to reaction mixture as an internal negative control for cleavage (lanes 2–4). Lane 1 contains total E. coli RNA. (b) Northern blot analysis of cleavage products. RNA probes complementary to 16S and 23S rRNA were used to detect fragmented rRNAs.