title: A Non-Stem-Loop CRISPR RNA Is Processed by Dual Binding Cas6.
abstract: A subclass of recently discovered CRISPR repeat RNA in bacteria contains minimally recognizable structural features that facilitate an unknown mechanism of recognition and processing by the Cas6 family of endoribonucleases. Cocrystal structures of Cas6 from Methanococcus maripaludis (MmCas6b) bound with its repeat RNA revealed a dual site binding structure and a cleavage site conformation poised for phosphodiester bond breakage. Two non-interacting MmCas6b bind to two separate AAYAA motifs within the same repeat, one distal and one adjacent to the cleavage site. This bound structure potentially competes with a stable but non-productive RNA structure. At the cleavage site, MmCas6b supplies a base pair mimic to stabilize a short 2 base pair stem immediately upstream of the scissile phosphate. Complementary biochemical analyses support the dual-AAYAA binding model and a critical role of the protein-RNA base pair mimic. Our results reveal a previously unknown method of processing non-stem-loop CRISPR RNA by Cas6.
authors.affiliation: Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: Yaming
authors.initials: Y
authors.lastname: Shao
authors.affiliation: Max-Planck-Institute for Terrestrial Microbiology, 35043 Marburg, Germany.
authors.forename: Hagen
authors.initials: H
authors.lastname: Richter
authors.affiliation: Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: Shengfang
authors.initials: S
authors.lastname: Sun
authors.affiliation: Department of Cellular Biochemistry, Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Kundan
authors.initials: K
authors.lastname: Sharma
authors.affiliation: Department of Cellular Biochemistry, Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: Max-Planck-Institute for Terrestrial Microbiology, 35043 Marburg, Germany; LOEWE Center for Synthetic Microbiology (Synmikro), 35043 Marburg, Germany.
authors.forename: Lennart
authors.initials: L
authors.lastname: Randau
authors.affiliation: Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA; Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA. Electronic address: hong.li@fsu.edu.
authors.forename: Hong
authors.initials: H
authors.lastname: Li
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism*; D017017:Methanococcus / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D019642:RNA, Archaeal / Q000737:chemistry / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D019642:RNA, Archaeal; D004722:Endoribonucleases
keywords: CRISPR RNA; crystal structures; endoribonucleases; protein-RNA interactions