Positive regulation of RpoS by small RNAs and the role of Hfq
Toby Soper 1, Pierre Mandin 2, Nadim Majdalani 2, Susan Gottesman 2, and Sarah A. Woodson 1
1: Johns Hopkins University, T. C. Jenkins Dept. of Biophysics
2: Laboratory of Molecular Biology, National Cancer Institute
Bacterial small non-coding RNAs (sRNAs) carry out both positive and negative
regulation of gene expression by pairing with mRNAs. This regulation often
requires the abundant Sm-like RNA chaperone protein Hfq. Three sRNAs, DsrA,
RprA, and ArcZ, positively regulate translation of the stationary phase sigma
factor RpoS, each pairing with the 5 leader to open up an inhibitory stem-loop
that prevents ribosome binding.
We previously showed that an (AAN)4 motif in the rpoS leader upstream of
the inhibitory stem is required for the RNA chaperone activity of Hfq. As
measured in vitro using native PAGE, rpoS leaders long enough to contain the
(AAN)4 repeat bind Hfq tightly and form ternary complexes with DsrA, RprA, and
ArcZ. Hfq strongly stabilized complexes of the long rpoS leader with DsrA at
25 deg. C and RprA and ArcZ at 37 deg. C. We show that the stabilities of the complexes
between the sRNAs and the rpoS leader correlated well with ability of the sRNAs
to activate rpoS::lacZ fusions in vivo. We also demonstrate the importance of the
(AAN)4 repeat motif for sRNA activity in vivo.
Hfq has separate binding sites for A-rich and U-rich RNA on opposite
sides of its homo-hexameric ring structure. However, we show that Hfq cobinding
to the two RNAs is insufficient to promote DsrA annealing to the rpoS
leader and that stable ternary complexes require RNARNA pairing. Rather, Hfq
acts by restructuring the rpoS leader to facilitate DsrA pairing.