Project Lecturer Kazuki Kato (Research Institute for Advanced Science and Technology, University of Tokyo) and his colleagues conducted research in collaboration with the Massachusetts Institute of Technology, showing that the Cas7-11-Csx29 complex, which is involved in a biological defense mechanism prokaryotes, is activated by binding to target RNA. We discovered that it is an RNA-dependent protease (protease) that cleaves the Csx30 protein. We also elucidated that the conformation of the Csx29 protein changes upon binding to the target RNA. It is expected to be applied to various technologies such as target RNA detection. The results were published in the online edition of the US scientific journal Science on November 3.
Cas7-11, a prokaryotic type III-E CRISPR-Cas enzyme that degrades viral RNA and DNA, forms a complex with a short RNA fragment (guide RNA) that has a sequence complementary to the target RNA, Acts as an RNA which is RNA dependent. cleavage enzyme (nuclease) that cleaves target RNA based on(Figure 1).
Type III-E CRISPR-Cas regions in prokaryotic genes produce five proteins, including Cas7-11 and Csx29 and Csx30, which are suggested to be involved in antiviral defense. Csx29 has an amino acid sequence similar to known proteases and was known to form a complex with Cas7-11, but its function was unknown.
Research found that Cas7-11 forms a complex with guide RNA and Csx29. We showed that when a target RNA with a sequence complementary to the guide RNA binds to the complex, Csx29 is activated and cleaves Csx30 in two, limiting its function.
In addition, using cryo-electron microscopy, the three-dimensional structures of the “Cas7-11-guide RNA-Csx29 complex” and the “Cas7-11-guide RNA-Csx29-target RNA complex” in which the target RNA is bound to him. are explained. he did These comparisons suggest that Csx29 undergoes conformational changes upon binding to target RNA and has Csx30 cleavage activity, revealing the molecular mechanism by which the Cas7-11-Csx29 complex acts as a nuclease that n RNA dependent.
Using the fact that Csx29 cleaves Csx30 only when there is a target RNA that is complementary to the guide RNA, we have also succeeded in detecting the presence of the target RNA as fluorescence. An RNA-dependent enzyme with dual properties of nuclease and protease is unprecedented and is expected to be applied to new technologies.
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