Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus

Raymond H J Staals; Yoshihiro Agari; Saori Maki-Yonekura; Yifan Zhu; David W Taylor; Esther van Duijn; Arjan Barendregt; Marnix Vlot; Jasper J Koehorst; Keiko Sakamoto; Akiko Masuda; Naoshi Dohmae; Peter J Schaap; Jennifer A Doudna; Albert J R Heck; Koji Yonekura; John van der Oost; Akeo Shinkai

doi:10.1016/j.molcel.2013.09.013

Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus

Mol Cell. 2013 Oct 10;52(1):135-145. doi: 10.1016/j.molcel.2013.09.013.

Authors

Raymond H J Staals^#¹, Yoshihiro Agari^#², Saori Maki-Yonekura^#², Yifan Zhu¹, David W Taylor^{3

4}, Esther van Duijn⁵, Arjan Barendregt⁵, Marnix Vlot¹, Jasper J Koehorst⁶, Keiko Sakamoto², Akiko Masuda⁷, Naoshi Dohmae⁷, Peter J Schaap⁶, Jennifer A Doudna^{4

8}, Albert J R Heck⁵, Koji Yonekura², John van der Oost¹, Akeo Shinkai²

Affiliations

¹ Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HB Wageningen, The Netherlands.
² RIKEN SPring-8 Center, 679-5148 Hyogo, Japan.
³ Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520-8114, USA.
⁴ California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158-2330, USA.
⁵ Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CH Utrecht, The Netherlands.
⁶ Laboratory of Systems and Synthetic Biology, Wageningen University, 6703 HB Wageningen, The Netherlands.
⁷ Global Research Cluster, RIKEN, Saitama 351-0198, Japan.
⁸ Department of Molecular and Cell Biology, and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720-3200, USA; Physical Biosciences Division, Lawrence Berkeley National Lab, Berkeley, CA 94720-3200, USA.

^# Contributed equally.

Abstract

The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Bacterial Proteins / metabolism*
CRISPR-Associated Proteins / chemistry
CRISPR-Associated Proteins / genetics
CRISPR-Associated Proteins / metabolism*
Clustered Regularly Interspaced Short Palindromic Repeats
High-Throughput Nucleotide Sequencing
Microscopy, Electron
Models, Molecular
Protein Conformation
Protein Subunits
RNA, Bacterial / chemistry
RNA, Bacterial / genetics
RNA, Bacterial / metabolism*
Ribonucleases / chemistry
Ribonucleases / genetics
Ribonucleases / metabolism*
Sequence Analysis, RNA
Spectrometry, Mass, Electrospray Ionization
Structure-Activity Relationship
Thermus thermophilus / genetics
Thermus thermophilus / metabolism*

Substances

Bacterial Proteins
CRISPR-Associated Proteins
Protein Subunits
RNA, Bacterial
Ribonucleases

Abstract

Publication types

MeSH terms

Substances

Grants and funding