c-di-GMP inhibits PlzA strand displacement activity in
vitro
Proteins other than RNA chaperones can accelerate RNA annealing in
vitro via indiscriminate mechanisms including molecular crowding and
shielding repulsive charge interactions between RNA molecules (82).
However, a hallmark of RNA chaperones is their ability to destabilize
dsRNA helices and displace one of the strands in the helix with another
RNA, termed strand displacement. Unlike RNA annealing, the melting and
strand displacement of stable, completely complementary dsRNA does not
occur without an RNA chaperone in vitro . We performed strand
displacement gel assays (68) to determine if PlzA has strand
displacement activity in vitro . Stable dsRNA (yellow band) was
formed and purified using the two unstructured fluorescent-labeled
complementary 21-nucleotide RNAs, J1 and M1, described above in Fig. 1.
The strand displacement assay was initiated by adding an excess of M1
unlabeled competitor RNA (uM1) with or without an RNA chaperone to the
J1M1 dsRNA; strand displacement activity is detected as a decrease in
dsRNA J1M1 (yellow band) and increases in J1uM1 dsRNA (red band) and M1
ssRNA (green band), as the unlabeled competitor RNA, uM1, replaces the
M1 RNA in the dsRNA (Fig. 2A). As noted, strand displacement did not
occur without an RNA chaperone (Fig. S2C, RNA only), while the known RNA
chaperone StpA had strong strand displacement activity (Fig. S2C, StpA).
Strand displacement was quantified using the ratio of J1M1 dsRNA (yellow
band) to total RNA and values graphed over time (Fig. 2B). We found that
PlzA had increased strand displacement activity and the amount of the
initial dsRNA at the endpoint of the assay was significantly lower
compared to the three negative controls, RNA only, c-di-GMP alone and
GrpE (Figs. 2B, 2C and S3B). However, the ability of PlzA to strand
displace was not as efficient as the positive control StpA in thein vitro assay. Furthermore, PlzA strand displacement activity
was modulated by c-di-GMP: the addition of c-di-GMP abrogated PlzA
strand displacement activity (Fig. 2B and C, compare green triangles and
blue inverted triangles). Inhibition of PlzA strand displacement
activity was specific to c-di-GMP as c-di-AMP had no effect (Fig. 2B,
compare green and orange triangles).
RNA-binding proteins affinity for RNA can be modulated by the binding of
metabolites, sRNAs, and proteins as well as by phosphorylation. There
are several RNA-binding proteins in B. subtilis , HutB, PyrR, and
TRAP, that are activated by the binding of histidine and
Mg2+, UMP and UTP, and tryptophan, respectively (83),
but there are no examples of RNA chaperone activity being regulated by a
second messenger. We hypothesized that the holo -PlzA may have a
lower affinity for dsRNA compared to apo -PlzA, thus bound
c-di-GMP would decrease strand displacement activity. We tested PlzA
affinity for dsRNA in the presence and absence of c-di-GMP by filter
binding assays, similar to Fig. 1D, using M1uJ1 dsRNA. The data
demonstrate c-di-GMP does not affect PlzA binding to double-stranded RNA
and the disassociation constants are nearly identical:KD of 61.7 ± 15.18 nM and 60.87 ± 8.45 nM with
and without c-di-GMP, respectively (Fig. 2D). In agreement with previous
reports, the KD of StpA was in the low µM range
(Fig. S4). Taken together, these data indicate that the binding of
c-di-GMP affects PlzA strand displacement activity but does not affect
the affinity of PlzA for ssRNA or dsRNA in vitro . In contrast to
other known metabolite-sensing RNA-binding proteins (83), RNA binding by
PlzA is not affected by c-di-GMP, but the RNA chaperone activity (strand
displacement) of PlzA is inhibited by c-di-GMP. To our knowledge, we
have identified the first RNA chaperone whose activity is regulated by a
second messenger.