PlzA has c-di-GMP-independent RNA annealing activity in
vitro
The role of PlzA in the enzootic cycle of B. burgdorferi has been
intensively studied (14,52,55-57), but the molecular basis for PlzA
function remains unknown. A comparative genomic analysis revealed PlzA
shares modest sequence similarity with ProQ (Fig. S1), an RNA chaperone
(22,76-79). The recently determined crystal structure revealed thatholo -PlzA has bilobed β-barrel domains connected by a short
linker (58). Several RNA-binding proteins, including cold shock proteins
A and E and eukaryotic Y-box proteins, utilize small β-barrels to
interact with RNA (16). We hypothesized that PlzA has RNA chaperone
activity, which could be modulated by the binding of c-di-GMP. To test
this hypothesis, we assayed the RNA annealing activity of PlzA in
vitro utilizing two artificial unstructured complementary 21-nucleotide
RNAs, termed J1 and M1, that were 5′ end-labeled with Cy5 or Cy3,
respectively (68). In vitro assays that employ artificial RNA
substrates are often used to test RNA chaperone activity. RNA chaperones
typically possess non-specific RNA chaperone activities even if they
have specific RNA targets. To assay “pure annealing” (without melting
internal base pairs from structured RNA), the J1 and M1 RNAs were
designed without internal secondary structure (68,71,80). The annealing
reactions were initiated by adding the M1 RNA to the J1 RNA, samples
were removed from the reaction over time, mixed with stop buffer and
resolved on a native polyacrylamide gel (Fig. 1A). The stop buffer
contains an excess of uM1 that binds to the remaining single-stranded
J1, effectively stopping the annealing reaction (Fig. 1A and S2A). RNA
annealing is visualized by a gel shift of the individual single-stranded
RNAs (ssRNAs) green and red bands to a yellow/orange band
double-stranded RNA (dsRNA) that migrates more slowly (Fig. S2B).
Annealing of complementary RNAs occurs unaided in vitro ; however,
addition of StpA, a protein with known RNA annealing activity (17,71),
significantly accelerated the base pairing (Fig. S2B). To measure the
rate of annealing, dsRNA formation was quantified using the ratio of
J1M1 dsRNA (yellow band) to total RNA over time (Fig. 1B). We found that
PlzA accelerated RNA annealing over time compared to the RNA only
reaction and the GrpE (which lacks RNA chaperone activity) and c-di-GMP
negative controls (Fig. 1B). Furthermore, the PlzA-mediated RNA
annealing rate was independent of c-di-GMP (Fig. 1B, compare green and
blue triangles). The amount of dsRNA formed by the endpoint of the assay
with PlzA was significantly higher than the negative controls, albeit
lower than the positive control (Figs. 1C and S3A).
We performed filter-binding assays to determine the binding affinity of
PlzA for the M1 ssRNA substrate (Fig. 1D). Cy3-labeled M1 RNA and serial
dilutions of PlzA proteins were incubated together, applied to a slot
blot apparatus, and filtered through two membranes (nitrocellulose and
positively charged nylon). The protein and protein-bound RNA bind to
nitrocellulose membrane, while unbound RNA flows through the
nitrocellulose and binds to the nylon membrane. The fraction of bound
RNA was calculated as a function of protein concentration (Fig. 1E). The
disassociation constant (K D) of StpA, the
non-specific positive control, was 838.5 ± 171.9 nM (Fig. S4), which
agrees with the K D determined previously (81).
Generally, non-specific interactions of RNA chaperones with RNA result
in K D in the low µM range, while specific
interactions are in the low nM range (18,21,68,80). TheK D of PlzA with and without c-di-GMP were 68.17 ±
7.77 nM and 93.37 ± 37.94 nM, respectively, which was not statistically
different (p = 0.4338), demonstrating that c-di-GMP does not
affect PlzA binding to single-stranded M1 (Fig. 1E).