Search

Prokaryotic type III CRISPR-Cas antiviral systems employ cyclic oligoadenylate (cA n ) signaling to activate a diverse range of auxiliary proteins that reinforce the CRISPR-Cas defense. Here we characterize a class of cA n -dependent effector proteins named CRISPR-Cas-associated messenger RNA (mRNA) interferase 1 (Cami1) consisting of a CRISPR-associated Rossmann fold sensor domain fused to winged helix-turn-helix and a RelE-family mRNA interferase domain. Upon activation by cyclic tetra-adenylate (cA 4 ), Cami1 cleaves mRNA exposed at the ribosomal A-site thereby depleting mRNA and leading to cell growth arrest. The structures of apo-Cami1 and the ribosome-bound Cami1-cA 4 complex delineate the conformational changes that lead to Cami1 activation and the mechanism of Cami1 binding to a bacterial ribosome, revealing unexpected parallels with eukaryotic ribosome-inactivating proteins.

CRISPR-associated Rossmann Fold (CARF) and SMODS-associated and fused to various effector domains (SAVED) are key components of cyclic oligonucleotide-based antiphage signaling systems (CBASS) that sense cyclic oligonucleotides and transmit the signal to an effector inducing cell dormancy or death. Most of the CARFs are components of a CBASS built into type III CRISPR-Cas systems, where the CARF domain binds cyclic oligoA (cOA) synthesized by Cas10 polymerase-cyclase and allosterically activates the effector, typically a promiscuous ribonuclease. Additionally, this signaling pathway includes a ring nuclease, often also a CARF domain (either the sensor itself or a specialized enzyme) that cleaves cOA and mitigates dormancy or death induction. We present a comprehensive census of CARF and SAVED domains in bacteria and archaea, and their sequence- and structure-based classification. There are 10 major families of CARF domains and multiple smaller groups that differ in structural features, association with distinct effectors, and presence or absence of the ring nuclease activity. By comparative genome analysis, we predict specific functions of CARF and SAVED domains and partition the CARF domains into those with both sensor and ring nuclease functions, and sensor-only ones. Several families of ring nucleases functionally associated with sensor-only CARF domains are also predicted., (Published by Oxford University Press on behalf of Nucleic Acids Research 2020.)

The analysis of ∼ 2000 bacterial genomes revealed that they all, without a single exception, encode one or more DNA polymerase III α-subunit (PolIIIα) homologs. Classified into C-family of DNA polymerases they come in two major forms, PolC and DnaE, related by ancient duplication. While PolC represents an evolutionary compact group, DnaE can be further subdivided into at least three groups (DnaE1-3). We performed an extensive analysis of various sequence, structure and surface properties of all four polymerase groups. Our analysis suggests a specific evolutionary pathway leading to PolC and DnaE from the last common ancestor and reveals important differences between extant polymerase groups. Among them, DnaE1 and PolC show the highest conservation of the analyzed properties. DnaE3 polymerases apparently represent an 'impaired' version of DnaE1. Nonessential DnaE2 polymerases, typical for oxygen-using bacteria with large GC-rich genomes, have a number of features in common with DnaE3 polymerases. The analysis of polymerase distribution in genomes revealed three major combinations: DnaE1 either alone or accompanied by one or more DnaE2s, PolC + DnaE3 and PolC + DnaE1. The first two combinations are present in Escherichia coli and Bacillus subtilis, respectively. The third one (PolC + DnaE1), found in Clostridia, represents a novel, so far experimentally uncharacterized, set.