In prokaryotes, the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-mediated defense system provides an RNA-based, adaptive immunity against bacteriophages and plasmids. The defense process is comprised of three key stages, including spacer acquisition, CRISPR RNA (crRNA) maturation and CRISPR interference. During the interference process, a surveillance complex, composed of CRISPR-associated (Cas) protein(s), is guided by a sequence-specific crRNA to target invading genetic elements for cleavage. The unique properties make CRISPR/Cas system a very valuable tool in several distinct areas, such as genomic manipulation, transcriptional regulation as well as the generation of phage-resistant strains for the industrial fermentation.
The CRISPR/Cas systems are categorized into the three major types (I, II and III). Although Csx3 protein is exclusively found in the Type III systems, Csx3 is not included into any of the Cas families and its function is largely unknown. In this study, we report the crystal structures of Archaeoglobus fulgidus Csx3 (AfCsx3) in free form, in complex with manganese ions and in complex with an RNA fragment at 2.95 Å, 3.0 Å and 2.9 Å, respectively. AfCsx3 harbors a ferredoxin-like fold and forms dimer both in the crystal and in solution. Our structure-based biochemical analysis demonstrates that the RNA binding sites and cleavage sites are located at two separate surfaces of the AfCsx3 dimer, suggesting a model to bind, tether and cleave the incoming RNA substrate. In addition, AfCsx3 displays robust 3’-deadenylase activity in the presence of manganese ions, which strongly suggests that AfCsx3 functions as a deadenylation exonuclease. Taken together, our results indicate that AfCsx3 is a Cas protein involved in RNA deadenylation and provide a framework for understanding the role of AfCsx3 in the Type III-B CRISPR/Cas system.
|School:||National University of Singapore (Singapore)|
|School Location:||Republic of Singapore|
|Source:||DAI-B 77/06(E), Dissertation Abstracts International|
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