PNPase physically associates with sRNAs and Hfq. (A) Northern blot analysis of RNAs that coimmunoprecipitated with affinity-tagged PNPase. PNPase-FLAG was immunoprecipitated from cell extracts prepared from late exponential phase cultures of a wild type (KR10000; WT) and derived strain expressing PNPase-FLAG (NRD1243; pnpFLAG) or the derived hfq deletion mutant (NRD1297; Δhfq pnpFLAG) using an anti-FLAG M2 agarose resin. For Western blots probed with anti-FLAG antibody, the ratio of protein samples from input and elution loaded was 1:1, and for Northern blots, the ratio of the volume of input to elution loaded was 1:162. SsrA and 5S rRNA are known not to be Hfq/PNPase dependent. (B) Fold enrichment for each RNA upon immunoprecipitation of PNPase. Fold enrichment of a given RNA upon immunoprecipitation was determined by first calculating the signal intensity per nanogram of RNA for the input and the elution from the Northern blots in A. The normalized elution signal was then divided by the input signal. (C) Western blot analysis of proteins that copurified with affinity-tagged PNPase. PNPase-FLAG was immunoprecipitated as described in A, except formaldehyde crosslinking was performed prior to purification and crosslinks were reversed post purification. Western blots were probed with antibodies against the 3xFLAG peptide, Hfq, β (RpoB), and β′ (RpoC).
