Thermus thermophilus Csm6 (TtCsm6) is a single-strand-specific endoribonuclease. (A) Multiple sequence alignment of Csm6 and Csx1 proteins from T. thermophilus (TtCsm6, GI:55978335), S. epidermidis (SeCsm6, GI:488416649), S. mutans (SmCsm6, GI:24379650), S. thermophilus (StCsm6, GI:585230687), and P. furiosus Csx1 (PfCsx1, GI:33359545), performed using ClustalOmega (McWilliam et al. 2013). HEPN domain active site residues are marked with asterisks. Secondary structure elements of TtCsm6 are indicated above the sequences. Disordered amino acid residues are indicated with dashed lines. (B) Nuclease activity assays performed with single- (ss) and double-stranded (ds) RNA and DNA oligonucleotide substrates. The 24-nt substrates have identical sequences and carry a Cy5 fluorophore group covalently attached to the 3′-terminus. Double-stranded substrates were prepared by annealing an unlabeled complementary strand to the respective single-stranded substrate. TtCsm6 (400 nM final concentration) was incubated with substrates (200 nM) at 37°C for 1 h. Cleavage products were analyzed by electrophoresis on a 15% denaturing polyacrylamide gel and detected using a fluorescence gel scanner. (C) Nuclease activity assays performed using TtCsm6 and identical ssRNA oligonucleotide substrates (24 nt) labeled with Cy5 at the 5′ or 3′ ends. The assay was performed as in B. A control digest with RNase T1 was used to generate RNA fragments of defined size, as indicated.
