Structural and biochemical characterization of in vivo assembled Lactococcus lactis CRISPR-Csm complex.

Autor: Sridhara S; Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306, USA.; Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, 40530, Sweden., Rai J; Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306, USA., Whyms C; Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 32306, USA., Goswami H; Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306, USA., He H; Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306, USA., Woodside W; Department of Microbiology, University of Georgia, Athens, GA, 30602, USA., Terns MP; Department of Microbiology, University of Georgia, Athens, GA, 30602, USA.; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA.; Department of Genetics, University of Georgia, Athens, GA, 30602, USA., Li H; Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306, USA. hong.li@fsu.edu.; Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 32306, USA. hong.li@fsu.edu.
Jazyk: angličtina
Zdroj: Communications biology [Commun Biol] 2022 Mar 29; Vol. 5 (1), pp. 279. Date of Electronic Publication: 2022 Mar 29.
DOI: 10.1038/s42003-022-03187-1
Abstrakt: The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle and is in addition regulated by cellular metabolites such as divalent metals and ATP. Recognition of the foreign or cognate target RNA (CTR) triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self or non-cognate target RNA (NTR) that differs from CTR only in its 3'-protospacer flanking sequence (3'-PFS). Here we employ electron cryomicroscopy (cryoEM), functional assays, and comparative cross-linking to study in vivo assembled mesophilic Lactococcus lactis Csm (LlCsm) at the three functional states: apo, the CTR- and the NTR-bound. Unlike previously studied Csm complexes, we observed binding of 3'-PFS to Csm in absence of bound ATP and analyzed the structures of the four RNA cleavage sites. Interestingly, comparative crosslinking results indicate a tightening of the Csm3-Csm4 interface as a result of CTR but not NTR binding, reflecting a possible role of protein dynamics change during activation.
(© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)
Databáze: MEDLINE
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