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RNA chaperones drive sRNA competition and target selection
Small non-coding RNAs must locate their proper targets among a myriad of spurious
candidates in the cell. The target search kinetics depends on co-localization, a search among
potential targets, and accurate recognition of the cognate target sequence. CRISPR guide
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RNAs and eukaryotic micro-RNAs are stably packaged with Cas and RISC protein,
respectively, which rapidly scan DNA or RNA targets while increasing the fidelity of target
site recognition (157). By contrast, overexpression experiments show that bacterial sRNAs
compete for a limited pool of Hfq hexamers in the cell (158, 159) by rapidly cycling on and
off the protein.
Initially, it was not clear how Hfq-bound RNAs exchange, given that sRNA and mRNA
containing U-rich or AAN recognition motifs have dissociation constants of 1–30 nM (160).
Fender and Wagner showed that the sRNA dissociation kinetics depends on the pool of free
RNA (161), and proposed that sRNAs actively displace each other from the proximal face of
the Hfq hexamer. RNA competition for the proximal face of Hfq was also observed in single
molecule FRET experiments (147). Regardless of the mechanism of sRNA exchange,
mRNAs are typically targeted within 1–2 minutes of sRNA induction. Rapid cycling is
needed to match sRNAs with their complementary targets within this time frame (158), and
to quickly retool regulatory circuits as growth conditions change (66).
Intrinsically disordered domains in RNA chaperones
In addition to structured RNA binding domains, RNA chaperones commonly contain
intrinsically disordered peptide (IDP) regions that are essential for their chaperone activity
(85). The frequent presence of IDPs in RNA chaperones has led to the “entropy transfer”
hypothesis, in which folding of the chaperone is coupled to increased disorder in the RNA
substrate (85, 162, 163). These disordered regions likely play different roles, depending on
their charge. Basic N-terminal polypeptides frequently increase the RNA affinity of the
chaperone and the ability to remodel RNA structures. For example, the basic N-terminal
domain of HIV NCp7 contributes substantially to acceleration of TAR hairpin annealing
with complementary DNA during minus strand transfer (164). The Mss116p and CYT-19
DEAD-box helicases possess an unstructured arginine-rich C-terminal extension that
recruits the helicase to the RNA substrate (165, 166) and may even help loosen the RNA
structure (167). Disordered regions may simply allow a chaperone to bind a variety of RNA
structures (168), as proposed for La protein (169). Polyamines or oligo-Lys also accelerate
refolding of misfolded RNA, however, suggesting that the distributed positive charge
associated with basic peptides lowers the energetic barrier for refolding (170, 171).
In contrast to the examples above, many DNA and RNA binding proteins contain acidic
peptides that mimic nucleic acid and inhibit nucleic acid binding to the core of the protein
(172–175). Recent experiments on Hfq illustrate the importance of these acidic peptides for
RNA chaperone activity (176, 177). The last 30 residues of
E. coli Hfq are disordered in
solution and protrude from the edge of the hexameric ring formed by the stable Sm domain
(178, 179). Acidic residues at the tip of the CTD, which are moderately conserved in
bacterial Hfq sequences, interact with the arginine patches on the rim of the hexamer (176),
displacing double-stranded RNA and inhibiting the binding of non-specific RNA and DNA
(177). In an analogous fashion, acidic residues at the C-terminus of NCp7 were also found to
increase the rate of dissociation from nucleic acid substrates (180).
Displacement of RNA by the CTD has several consequences for Hfq’s role in sRNA
regulation. First, the CTD prevents separation of sRNA-mRNA strands (the reverse of
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annealing) while recycling Hfq to bind another RNA. Second, it creates a hierarchy of sRNA
regulation because the CTDs do not displace AAN RNA from the distal face of Hfq,
enabling RNAs containing AAN motifs to outcompete other sRNAs for access to Hfq in the
cell (177). Because the length of the CTD and number of acidic residues vary among
bacterial species, these features may be used to fine-tune the permissiveness and turnover
rate of Hfq in different organisms (181).
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