The Amyloid Region of Hfq Riboregulator Promotes DsrA:rpoS RNAs Annealing

Abstract

Hfq is a bacterial RNA chaperone which promotes the pairing of small noncoding RNAs to target mRNAs, allowing post-transcriptional regulation. This RNA annealing activity has been attributed for years to the N-terminal region of the protein that forms a toroidal structure with a typical Sm-fold. Nevertheless, many Hfqs, including that of Escherichia coli, have a C-terminal region with unclear functions. Here we use a biophysical approach, Synchrotron Radiation Circular Dichroism (SRCD), to probe the interaction of the E. coli Hfq C-terminal amyloid region with RNA and its effect on RNA annealing. This C-terminal region of Hfq, which has been described to be dispensable for sRNA:mRNA annealing, has an unexpected and significant effect on this activity. The functional consequences of this novel property of the amyloid region of Hfq in relation to physiological stress are discussed.

Synchrotron Radiation Circular Dichroism, a New Tool to Probe Interactions between Nucleic Acids Involved in the Control of ColE1-Type Plasmid Replication

“…Before use, the Hfq-CTR peptide was reconstituted in water at 20 mg/mL. We determined previously that pH 5 was the most appropriate for the formation of the complex with nucleic acids [24]. We also chose to avoid the addition of salts (except those already present in DNA, protein and peptide solutions) to allow better investigation of the conditions in deep UV [34,35].…”

“…We also chose to avoid the addition of salts (except those already present in DNA, protein and peptide solutions) to allow better investigation of the conditions in deep UV [34,35]. Previously, we ensured that the presence of salts (NaCl 50 mM) does not significantly change the activity of Hfq-CTR [24].…”

“…The effect of Hfq-CTR was analyzed at 15 • C to minimize spontaneous annealing between nucleic acids that could occur [24]. For kinetic measurements, an apparent catalytic kinetic constant k cat app was determined during initial rate conditions.…”

“…Melting of dsDNA is mandatory for the replication process, as it is not achieved by the DNA polymerase. SRCD makes it possible to follow the transition from double-to single-strand DNA, as well as the reverse transition [24,42]. Indeed, the spectral region between 320 and 170 nm contains several electronic transitions of interest.…”

“…It is believed that the NTR is crucial for the interactions of Hfq with RNAs [21][22][23]. On the other hand, the latter domain is 30-35 aa-long and participates in the functions of Hfq required to facilitate mRNA-small RNA interactions [19,24]. Intriguingly, the CTR can form an amyloidlike structure [25] that can bind to DNA, making the DNA molecule more compact and condensed [26][27][28].…”