A General Mechanism for the General Stress Response in Bacteria.

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. These results additionally resolve the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases, revealing a common coiled-coil linker transduction mechanism. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

Metal Dependence and Functional Diversity of Type I Cas3 Nucleases.

Type I CRISPR-Cas systems provide prokaryotes with protection from parasitic genetic elements by cleaving foreign DNA. In addition, they impact bacterial physiology by regulating pathogenicity and virulence, making them key players in adaptability and evolution. The signature nuclease Cas3 is a phosphodiesterase belonging to the HD-domain metalloprotein superfamily. By directing specific metal incorporation, we map a promiscuous metal ion cofactor profile for Cas3 from Thermobifida fusca (Tf). Tf Cas3 affords significant ssDNA cleavage with four homo-dimetal centers (Fe2+, Co2+, Mn2+, and Ni2+), while the diferrous form is the most active and likely biologically relevant in vivo. Electron paramagnetic resonance (EPR) spectroscopy and Mössbauer spectroscopy show that the diiron cofactor can access three redox forms, while the diferrous form can be readily obtained with mild reductants. We further employ EPR and Mössbauer on Fe-enriched proteins to establish that Cas3″ enzymes harbor a dinuclear cofactor, which was not previously confirmed. We demonstrate that the ancillary His ligand is critical for efficient ssDNA cleavage but not for diiron assembly or small molecule hydrolysis. We further explore the ability of Cas3 to hydrolyze cyclic mononucleotides and show that Tf Cas3 hydrolyzes 2'3'-cAMP with catalytic efficiency comparable to that of the conserved virulence factor A (CvfA), an HD-domain protein hydrolyzing 2'3'-cylic phosphodiester bonds at RNA 3'-termini. Because this CvfA activity is linked to virulence regulation, Cas3 may also utilize 2'3'-cAMP hydrolysis as a possible molecular route to control virulence.

Telomere DNA G-quadruplex folding within actively extending human telomerase.

Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K+ versus GQ-destabilizing Li+ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase-DNA complex. Addition of the telomerase processivity factor POT1-TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li+ cations. This result suggests GQ folding synergizes with POT1-TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K+ but not Li+, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.