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. 2016 Nov 11;36(3):374–387. doi: 10.15252/embj.201694639

Figure EV1. C‐terminally tagged RNase E is functional and facilitates purification of labelled RNA–RNase E complexes.

Figure EV1

  1. RNase E is required for processing of 9S rRNA into mature 5S rRNA, and we assessed 5S rRNA processing in our His‐FLAG‐tagged RNase E strain. Enterohaemorrhagic E. coli (EHEC) O157:H7 str. Sakai and the isogenic rne‐HTF insertion mutant were grown to an OD600 of 0.6 in LB broth at 37°C and total RNA was harvested. To compare wild‐type and impaired 5S rRNA processing, we additionally cultured E. coli str. K12 (N3433) and the isogenic temperature‐sensitive rne‐3071 mutant (N3431) to OD600 0.6. The rne‐3071 mutant was temperature‐shifted to 43°C for 30 min and total RNA harvested. 1 μg of total RNA was separated on an 8% TBE–urea polyacrylamide gel and blotted for 9S rRNA. Mature 5S rRNA is shown in the SYBR‐stained loading control below.
  2. RNA–protein complexes were eluted from Ni‐NTA resin (see Materials and Methods) and precipitated and 50% of the eluate separated on a NuPAGE Bis‐Tris 4–12% gel and silver‐stained. E. coli O157:H7 str. Sakai (untagged; negative control) and the isogenic rne‐HTF mutant were analysed for stringency of the dual‐affinity purification.
  3. Replicate [32P]‐labelled RNA–RNase E complexes were separated on NuPAGE Bis‐Tris 4–12% gradient gels and transferred to nitrocellulose membranes. The labelled complexes were imaged by autoradiography and complexes with a molecular mass equivalent to RNase E–RNA fragments (dashed boxes) were excised from the membrane (see Materials and Methods).

Source data are available online for this figure.