A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats.
Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.
Beloglazova
Natalia
N
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.
Brown
Greg
G
Zimmerman
Matthew D
MD
Proudfoot
Michael
M
Makarova
Kira S
KS
Kudritska
Marina
M
Kochinyan
Samvel
S
Wang
Shuren
S
Chruszcz
Maksymilian
M
Minor
Wladek
W
Koonin
Eugene V
EV
Edwards
Aled M
AM
Savchenko
Alexei
A
Yakunin
Alexander F
AF
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D012313:RNA; D004722:Endoribonucleases