93 Structures guided insight into mechanisms of RNA-guided surveillance of foreign DNA

Abstract

Viruses that infect bacteria are the most diverse and abundant biological agents on the planet, causing roughly 1023 infections every second. In response to these pervasive viral predators, bacterial have evolved a diverse repertoire of anti-viral defense mechanisms. Adaptive immune systems in bacteria rely on a repetitive chromosomal locus called a CRISPR (clusters of regularly interspaced short palindromic repeats) and a diverse set of CRISPR-associated (Cas) proteins. The adaptive immune system in Escherichia coli K12 consists of eight cas genes and a downstream CRISPR. The CRISPR locus is transcribed and processed into mature 61-nt CRISPR-derived RNAs (crRNAs) that assembles with 11 Cas proteins into a 405 kDa multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent X-ray and cryo-EM structures of Cascade reveal conformational rearrangements upon binding target DNA. These models provide valuable snap shots of the surveillance machine before and after target binding (Hochstrasser et al., 2014; Jackson et al., 2014; Wiedenheft et al., 2011; Zhao et al., 2014). However, current models do not explain how Cascade distinguishes a complementary target in the host CRISPR locus (i.e. self DNA) form identical sequences in the genome of a genetic invader (i.e. non-self DNA). Here we integrate existing models with new structures of Cascade in a molecular dynamic simulation that reveals functional roles for conserved regions of the complex that are critical for target recognition and recruitment of a transacting nuclease that is required for target degradation.