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).