Gel shift experiments with CCA-adding enzyme, Hfq and BSA. (A) Only Hfq (either alone or in the presence of CCA-adding enzyme) was binding to the radioactively labeled tRNA substrate (without CCA terminus), leading to a reduced electrophoretic mobility on a native polyacrylamide gel (arrow). The identical band shifts in the gel after sample preincubation for 1 and 10 minutes indicate that the binding equilibrium was reached within 1 minute and that the Hfq-tRNA interaction is rather stable over time. (B) The tRNA/Hfq complex was competed by a nonspecific plasmid run-off transcript (left) or a transcript corresponding to the 3'-end of rpsO mRNA. While the rpsO RNA (carrying an Hfq binding site) efficiently replaced the bound tRNA at concentrations above 100 fmol (> 5-fold excess), the plasmid transcript could not compete for binding at any concentration, indicating a specific interaction of Hfq with the tRNA.