Structure and Function of an in vivo Assembled Type III-A CRISPR-Cas Complex Reveal Critical Roles of Dynamics in Activity Control

Abstract

The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle, but its molecular basis for regulation remains unexplained. Recognition of the foreign RNA, or cognate target RNA (CTR), triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self-RNA, or noncognate target RNA (NTR) that differs from CTR only in its 3-protospacer flanking sequence. Here we captured four structures of in vivo assembled Lactococcus lactis Csm (LlCsm) by electron cryomicroscopy representing both the active and the inactive states. Surprisingly, in absence of bound RNA, LlCsm largely forms a minimal assembly lacking the Csm2 subunit with a stably bound catalytic subunit Csm1. Comparison of the minimal LlCsm structure and activities, both in vitro and in vivo, with those of fully assembled LlCsm reveals a molecular mechanism responsible for the viral RNA-activated and self RNA-inhibited activity of Csm1 through protein dynamics. Graphic Art Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/428455v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e9c428org.highwire.dtl.DTLVardef@4d3397org.highwire.dtl.DTLVardef@76184dorg.highwire.dtl.DTLVardef@1359dd5_HPS_FORMAT_FIGEXP M_FIG C_FIG