Figure 8.

ChiX positively regulates IS 10 transposase translation.

A. hfq⁺ cells containing a chromosomal IS 10 _1–339‐lac Z translational fusion were transformed with a plasmid expressing ChiX (pDH765), SgrS (pDH764) or a vector control (pDH763). Transformants were grown to mid‐exponential phase in LB media and β‐galactosidase activity was measured. Error bars show the standard error of the mean for two independent experiments (n = 7) and the relative expression is shown above the graph, where transposase‐lacZ expression in the presence of vector was set to 1. RNA was extracted immediately before the Miller assay.

B. A 3.5 μg of total RNA (three biological isolates) was used for a Northern blot using a 5′³²P‐labeled oligonucleotide (SgrS) or internally labeled antisense RNA probe (ChiX, 5S rRNA).

C. Primer extension analysis of 10 μg of total RNA (four biological isolates) was used to detect RNA‐IN‐lac Z transcript and lpp was analyzed as an internal control. The ratio of IN‐lac Z:lpp was normalized to the vector control and is shown with standard error of the mean as a graph above the gel images.

D. 5′³²P labeled ChiX RNA (100 nM) was incubated with purified Hfq or Hfq and in vitro transcribed RNA‐IN before limited cleavage with Pb²⁺. An RNase T1 sequencing lane (G; lane 1) and untreated controls (lanes 2–4) are shown. An aliquot of binding reactions was analyzed on a 6% native polyacrylamide gel (bottom panel). The secondary structure of ChiX is shown (right panel) with three Hfq‐binding sites (Hfq‐I/II/III) highlighted in red.