Type III CRISPR complexes from Thermus thermophilus.
Pathogen-specific acquired immunity in bacteria is mediated by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems. Thermus thermophilus strain HB8 contains CRISPR systems of several major subtypes (type I, IIIA and IIIB), and has become a widely studied model for CRISPR biology. We have selected two highly expressed CRISPR spacers, crRNA 2.1 and crRNA 2.2, and have enriched endogenous T. thermophilus proteins that co-purify with these crRNAs. Mass spectroscopy indicates that the chromatography protocol enriches predominantly Csm complex subunits, but also Cmr subunits. After several chromatographic steps, size exclusion chromatography indicated a molecular mass of the crRNA associated complex of 265±69 kDa. In agreement with earlier work, crRNAs of different lengths (containing the selected spacers) were observed. Most of these were completely lost when several T. thermophilus csm genes were ablated.
International Institute of Molecular and Cell Biology, Warsaw, Poland; Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Łódź, Poland.
Marta
M
Szychowska
International Institute of Molecular and Cell Biology, Warsaw, Poland.
Wojciech
W
Siwek
International Institute of Molecular and Cell Biology, Warsaw, Poland.
Damian
D
Pawolski
International Institute of Molecular and Cell Biology, Warsaw, Poland.
Asgar Abbas
AA
Kazrani
Microbiology Department, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland.
Krzysztof
K
Pyrc
International Institute of Molecular and Cell Biology, Warsaw, Poland; Institute of Biochemistry and Biophysics PAS, Warsaw, Poland.
Matthias
M
Bochtler
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005798:Genes, Bacterial; D008970:Molecular Weight; D012329:RNA, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D016963:Thermus thermophilus / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D004720:Endonucleases