Fig. 1.

The Csy proteins assemble into a large ribonucleoprotein complex. (A) Two CRISPR loci flank a set of cas genes in the P. aeruginosa (PA14) genome. Each CRISPR consists of a series of direct repeats (black hexagons) that are separated by unique spacer sequences (blue cylinders). Both CRISPRs are flanked by a leader sequence (black arrow). Dyad symmetry within each direct repeat results in a CRISPR RNA transcript consisting of a series of hairpins (black) that are recognized by Csy4 (cyan oval). Each repeat is separated by unique spacer sequences (dashed blue line). (B) Coomassie-blue stained SDS-polyacrylamide gel of the affinity purified Csy complexes (Upper). An N-terminal His-tags (*) on Csy4 can be used as bait to pull down the other untagged Csy proteins. The His-tag is removable by treating with TEV protease (right lane). Denaturing polyacrylamide gel of phenol extracted crRNAs isolated from the Csy complexes (Lower). The N-terminal His-tag does not interfere with particle assembly or CRISPR RNA processing. (C) Native mass spectrum of the Csy complex. The intact Csy complex has a total molecular weight of 350.4 kDa (purple triangles) with a subunit stoichiometry corresponding to Csy1₁∶Csy2₁∶Csy3₆∶Csy4₁∶crRNA₁. A complex with a slightly higher mass (352.5 kDa) is also observed. The additional mass in this complex is due to incomplete removal of the His-tag from the Csy4 subunit following digestion with the TEV protease (blue triangles). A complex lacking Csy1 and Csy2 was also identified (pink triangles). At the low m/z region of the spectrum free Csy4 (orange triangle) and a complex of Csy4 and crRNA (green triangles) are observed. For each distribution the charge state of the major peak is given.