TY - JOUR
T1 - Target sequence requirements of a type III-B CRISPR-Cas immune system
AU - Johnson, Kaitlin
AU - Learn, Brian A.
AU - Estrella, Michael A.
AU - Bailey, Scott
N1 - Funding Information:
This work was supported by National Institutes of Health Grants GM097330 (to S. B.) and Ruth L. Kirschstein National Institutes of Health F31 fellowship GM105364 (to M. A. E.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2019 Johnson et al.
PY - 2019/6/28
Y1 - 2019/6/28
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.RA119.008728
DO - 10.1074/jbc.RA119.008728
M3 - Article
C2 - 31110048
AN - SCOPUS:85068662032
SN - 0021-9258
VL - 294
SP - 10290
EP - 10299
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -