Figure 4.

Deletion of the Hfq C-terminus results in a decreased RNA annealing activity and abolishes the ability to bind two RNAs simultaneously. (A) Annealing of two fluorophore-labelled RNA 21-mers can be accelerated by RNA chaperones (top; 50) that can be monitored by FRET. Bottom, 5 nM of each RNA oligonucleotide Cy5-21R⁺ and Cy3-21R⁻ were annealed at 37°C either in the absence or in the presence of 1 µM Hfq_wt or Hfq₆₅ hexamer. FRET was calculated as ratio of acceptor/donor emission (F_Cy5/F_Cy3), normalized to 1 at t_180s, and the data was fitted with the second-order reaction equation for equimolar initial reactant concentrations y = A [1 − 1/(k_ann t + 1)]. Hfq_wt and Hfq₆₅ increased the observed annealing reaction constant k_ann 7- and 2-fold, respectively. (B) In a set-up with the non-complementary Cy5-21R⁺ and Cy3-21R⁺ (Cy3-duplex) RNA oligonucleotides, simultaneous binding of the two RNAs to a protein can be measured (top). Bottom, the time-resolved FRET index curves were fitted with y = A [1 − 1/(k_db t + 1)] to yield the dual RNA oligonucleotide binding rate constant k_db. The RNA oligonucleotides by themselves do not anneal, whereas Hfq_wt can bind both RNAs simultaneously. Incubation with Hfq₆₅ resulted in a k_db 10-fold lower than with Hfq_wt, indicating that the C-terminal truncation coincides with a severely reduced capacity for simultaneous binding of the two non-complementary RNA oligonucleotides. The FRET index in this graph was not normalized to indicate the reaction amplitudes.