Fig. 6

The rlmH::ISS1 ribosomes bind poorly to intron RNA. a. In vitro gel-shift assays show that rlmH::ISS1 mutant 70S ribosomes are defective in binding to intron RNA. Gels show wild-type (WT) and rlmH::ISS1 ribosome-intron binding, where substrate (S) is radiolabeled in vitro-transcribed intron substrate, with bound product (P) represented higher in the gel. Increasing concentrations of ribosomes are shown with values across the top. Multiple bound products likely represent various ribosome-bound intron RNA conformations [16]. Below is a binding curve based on quantifying the intensity of the bands in these gels, with the average fraction bound plotted against the concentration of ribosomes. b. In vivo intron pull-down is consistent with rlmH::ISS1 ribosome defect in binding intron RNA. The pull-down construct is expressed within small exons (E1 and E2, green), with the intron encoded protein (IEP, gray) expressed in trans. Intron RNA containing a streptavidin aptamer (SA, black) was captured using streptavidin resin (1), and the RNA was subjected to a Northern blot (2), probing for intron RNA and 16S rRNA. The ratio of rRNA per intron RNA was plotted, with individual replicates as data points, the mean as a horizontal line, and standard deviation as whiskers. Significantly more rRNA (p < 0.05) copurified with intron RNA in the control strain than the rlmH::ISS1 mutant strain. Full blot is in Additional File 1: Fig. S18