Two Seemingly Homologous Noncoding RNAs Act Hierarchically to Activate glmS mRNA Translation
Figure 4
GlmZ, Not GlmY, Enhances glmS Translation In Vitro
(A) In vitro synthesized glmS::gfp fusion mRNA (0.1 μM) was translated with reconstituted 70S ribosomes in the absence (lanes 1–6) or presence (lanes 7–12) of Hfq (0.5 μM) and absence (lanes 1–3 and 10–12) or presence (lanes 4–9) of GlmZ RNA (1 μM). Protein samples were prepared upon incubation for 10, 20, or 30 min, and subjected to western blot analysis using monoclonal α-GFP antibodies to detect the synthesized GlmS::GFP fusion protein.
(B) In vitro translation of glmS::gfp mRNA (0.1 μM) in the presence of Hfq (0.5 μM) for 15 min. Reactions contained increasing amounts of GlmZ RNA (0, 0.1, 0.2, 0.5, and 1 μM; lanes 1–5), GlmY RNA (0.1, 0.2, 0.5, and 1 μM; lanes 7–10), the 3′ truncated GlmZ153 RNA (1 μM; lane 6), or 0.1 μM GlmZ RNA with increasing amouts of GlmY RNA (0.1, 0.2, and 0.5 μM; lanes 11–13). Note that with fixed GlmZ and increasing GlmY concentrations, GlmS synthesis actually decreases (lanes 11–13). This may be explained by competition of GlmY with GlmZ for Hfq-binding, which would inhibit GlmZ-glmS mRNA pairing and, consequently, GlmS synthesis.