Molecular characterisation of two novel double-stranded RNA elements from Phlebiopsis gigantea.

The incomplete sequences of two large, 10-12 kbp, double-stranded RNAs (dsRNAs) found in the TW-2 isolate of the saprophytic fungus, Phlebiopsis gigantea (Pg) are reported. Both PgV-TW2 dsRNA1 and dsRNA2 potentially encode fusion proteins which are apparently expressed by a translational frameshifting mechanism. The C-terminal region of both predicted proteins was 21% identical and contained the eight motifs conserved in RNA-dependent RNA polymerases of dsRNA mycoviruses and had highest similarity with members of the family Totiviridae, but possibly do not form virions. The remainder of the N-terminal protein sequences predicted from the PgV-TW2 dsRNA1 and dsRNA2 sequences and the 3'-terminal nucleotide sequences of both dsRNAs had no homology with one another or any sequence in the database suggesting that individually both may be members of novel families of mycoviruses.

Sensor I threonine of the AAA+ ATPase transcriptional activator PspF is involved in coupling nucleotide triphosphate hydrolysis to the restructuring of sigma 54-RNA polymerase.

Transcriptional initiation invariably involves the transition from a closed RNA polymerase (RNAP) promoter complex to a transcriptional competent open complex. Activators of the bacterial sigma(54)-RNAP are AAA+ proteins that couple ATP hydrolysis to restructure the sigma(54)-RNAP promoter complex. Structures of the sigma(54) activator PspF AAA+ domain (PspF(1-275)) bound to sigma(54) show two loop structures proximal to sigma(54) as follows: the sigma(54) contacting the GAFTGA loop 1 structure and loop 2 that classifies sigma(54) activators as pre-sensor 1 beta-hairpin AAA+ proteins. We report activities for PspF(1-275) mutated in the AAA+ conserved sensor I threonine/asparagine motif (PspF(1-275)(T148A), PspF(1-275)(N149A), and PspF(1-275)(N149S)) within the second region of homology. We show that sensor I asparagine plays a direct role in ATP hydrolysis. However, low hydrolysis rates are sufficient for functional output in vitro. In contrast, PspF(1-275)(T148A) has severe defects at the distinct step of sigma(54) promoter restructuring. This defect is not because of the failure of PspF(1-275)(T148A) to stably engage with the closed sigma(54) promoter, indicating (i) an important role in ATP hydrolysis-associated motions during energy coupling for remodeling and (ii) distinguishing PspF(1-275)(T148A) from PspF(1-275) variants involved in signaling to the GAFTGA loop 1, which fail to stably engage with the promoter. Activities of loop 2 PspF(1-275) variants are similar to those of PspF(1-275)(T148A) suggesting a functional signaling link between Thr(148) and loop 2. In PspF(1-275) this link relies on the conserved nucleotide state-dependent interaction between the Walker B residue Glu(108) and Thr(148). We propose that hydrolysis is relayed via Thr(148) to loop 2 creating motions that provide mechanical force to the GAFTGA loop 1 that contacts sigma(54).