Browsing by Author "Rowe, Sebastian J."

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    Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex
    (Cold Spring Harbor Lab Press, 2019) Nasef, Mohamed; Muffly, Mary C.; Beckman, Andrew B.; Rowe, Sebastian J.; Walker, Forrest C.; Hatoum-Aslan, Asma; Dunkle, Jack A.; University of Alabama Tuscaloosa
    CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that Type III CRISPR-Cas systems synthesize second messenger molecules previously unknown to exist in prokaryotes, cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal Type III systems, cOA generation and its regulation have not been explored for the Staphylococcus epidermidis Type III-A CRISPR-Cas system, a longstanding model for CRISPR-Cas function. Here, we demonstrate that this system performs Mg2+-dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest a natural mechanism to dampen immunity once the foreign RNA is destroyed.
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    Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition
    (Elsevier, 2020) Rowe, Sebastian J.; Mecaskey, Ryan J.; Nasef, Mohamed; Talton, Rachel C.; Sharkey, Rory E.; Halliday, Joshua C.; Dunkle, Jack A.; University of Alabama Tuscaloosa
    Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m(6) (2)A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity-binding assays. We demonstrate that residues in alpha 4, alpha 5, and the alpha 5-alpha 6 linker are essential for methyltransferase function, including an aromatic residue on alpha 4 that likely forms stacking interactions with the substrate adenosine and basic residues in alpha 5 and the alpha 5-alpha 6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.