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.