The CRISPR system : a new frontier in prokaryotic molecular biology

1. Structure and function of archaeal CASCADE.
Systematic co-expression of CRISPR proteins (Objective 1b) led to the identification of a stable interaction between Cas5 and Cas7. We recognised this pair as the core of the CASCADE complex for viral DNA targetting in archaea. In collaboration with the Lawrence lab (Montana), we reported the first structure of the widely conserved Cas7 protein, revealing an RRM fold related to that found in other CAS proteins. This helped to simplify our understanding of the evolution of the CRISPR System. We showed that Cas7 has an important role in crRNA binding, probably forming the backbone of the CASCADE complex. Further, we identified a third interacting subunit of the complex, Csa5, and characterised the bound crRNA (Objective 2a). This work was published in JBC in 2011.
2. Structure and mechanism of the CMR complex (Objective 2d).
CMR is the only known prokaryotic RNA silencing machinery and as such has been the subject of intense interest. The 7-subunit CMR complex for crRNA-mediated RNA degradation was purified to homogeneity from S. solfataricus. Tagged versions were constructed and expressed, allowing targetted mutation of subunits. In vitro, the complex had the expected RNA targetting activity but with a number of important differences from the previously published Pyrococcus complex. In particular, we demonstrated that the endonuclease activity was sequence dependent and that both target and guide RNA could be cleaved. Structural studies revealed the structure of the Cmr7 subunit and an EM reconstruction of the entire 400 kDa complex (collaboration with Laura Spagnolo, Edinburgh). This work was published in Molecular Cell (online 5th Jan 2012).