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J Biol Chem. 2019 Jun 28;294(26):10290-10299. doi: 10.1074/jbc.RA119.008728. Epub 2019 May 19.

Target sequence requirements of a type III-B CRISPR-Cas immune system.

Author information

1
From the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health and.
2
From the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health and scott.bailey@jhu.edu.
3
Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205.

Abstract

CRISPR-Cas systems are RNA-based immune systems that protect many prokaryotes from invasion by viruses and plasmids. Type III CRISPR systems are unique, as their targeting mechanism requires target transcription. Upon transcript binding, DNA cleavage by type III effector complexes is activated. Type III systems must differentiate between invader and native transcripts to prevent autoimmunity. Transcript origin is dictated by the sequence that flanks the 3' end of the RNA target site (called the PFS). However, how the PFS is recognized may vary among different type III systems. Here, using purified proteins and in vitro assays, we define how the type III-B effector from the hyperthermophilic bacterium Thermotoga maritima discriminates between native and invader transcripts. We show that native transcripts are recognized by base pairing at positions -2 to -5 of the PFS and by a guanine at position -1, which is not recognized by base pairing. We also show that mismatches with the RNA target are highly tolerated in this system, except for those nucleotides adjacent to the PFS. These findings define the target requirement for the type III-B system from T. maritima and provide a framework for understanding the target requirements of type III systems as a whole.

KEYWORDS:

CRISPR-Cas; CRISPR-RNA; Cmr effector complex; DNA; RNA; bacterial immunity; crRNA; deoxyribonuclease (DNase); nucleotide mismatch; ribonuclease; sequence similarity

PMID:
31110048
DOI:
10.1074/jbc.RA119.008728

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