During the prokaryotic type III CRISPR-Cas immune response, infection triggers the production of cyclic oligoadenylates, which bind and activate CARF domain-containing proteins 1 , 2 . Many type III loci are associated with proteins in which the CARF domain is fused to an endonuclease-like domain 3 , 4 ; however, with the exception of the well-characterized Csm6/Csx1 RNases 5 , 6 , whether and how these inducible effectors provide defense is not known. Here we investigated one of such type III CRISPR accessory proteins, Card1. Card1 forms a symmetrical dimer with a large central cavity between its CARF and restriction endonuclease (REase) domains that binds cA 4 . Ligand binding results in a conformational change where individual monomers rotate relative to each other to form a more compact dimeric scaffold wherein a Mn cation coordinates to the catalytic residues and activates the cleavage of single, but not double, stranded nucleic acids (DNA and RNA). In vivo , Card1 activation induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used by CRISPR systems to provide immunity.

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Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger elemental pausing by stabilizing a half-translocated (RNA-not-DNA) state and by promoting RNAP swiveling that other factors can enhance. The universal transcription factor NusG (Spt5 in eukaryotes and archaea) N-terminal domain (NGN) modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs (e.g.,Bacillus subtilis) enhance some pauses whereas anti-pausing NusGs (e.g.,Escherichia coli) suppress some pauses. Little is known about pausing and NusG in the human pathogenMycobacterium tuberculosis (Mtb). Using biochemistry and cryo-electron microscopy, we show thatMtbNusG is a pro-pausing NusG that captures paused, swiveled RNAP by contacts to the RNAP protrusion and to a nontemplate strand–DNA wedge inserted between the NGN and the RNAP gate loop. On the other hand, we find that anti-pausingE. coliNGN contacts the RNAP gate loop to inhibit swiveling and pausing ofMtbRNAP. Using CRISPR-mediated mycobacterial genetics, we show that a pro-pausing NGN is required to support robust mycobacterial growth. Our results define an essential function of NusG in mycobacteria and the structural basis of pro-vs. anti-pausing NusG activity with broad implications for NusG function in all domains of life.

The Clp protease system is a promising, noncanonical drug target against Mycobacterium tuberculosis (Mtb). Unlike in Escherichia coli , the Mtb Clp protease consists of two distinct proteolytic subunits, ClpP1 and ClpP2, which hydrolyze substrates delivered by the chaperones ClpX and ClpC1. While biochemical approaches uncovered unique aspects of Mtb Clp enzymology, its essentiality complicates in vivo studies. To address this gap, we leveraged new genetic tools to mechanistically interrogate the in vivo essentiality of the Mtb Clp protease. While validating some aspects of the biochemical model, we unexpectedly found that only the proteolytic activity of ClpP1, but not of ClpP2, is essential for substrate degradation and Mtb growth and infection. Our observations not only support a revised model of Mtb Clp biology, where ClpP2 scaffolds chaperone binding while ClpP1 provides the essential proteolytic activity of the complex; they also have important implications for the ongoing development of inhibitors toward this emerging therapeutic target.