Investigation of the RNA-Protein Structure-Function Relationships in the CRISPR-Cas10 Complex and Erythromycin-Resistance RNA Methyltransferases

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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, known as 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 Gram-positive bacterium, Staphylococcus epidermidis, as well as the specific target RNA sequence requirements. In chapter 2, 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 synthesis is antagonized by Csm3-mediated target RNA cleavage. In chapter 3, we mechanistically investigated the effect of single and multiple mismatches on the activation of cOA synthesis of type III-A Cas10-Csm complex from S. epidermidis. We show that mismatches at specific positions in target RNA significantly reduced the amount of cOAs produced in a sequence context-dependent manner. We also show that the defects are not due to perturbations in binding efficiencies or in RNA cleavage.In chapter 4, we focus on an erythromycin resistance methyltransferases (Erm) found in many Gram-positive pathogens. Erm proteins are S-adenosyl methionine-dependent Rossmann-fold methyltransferases which convert A2058 of 23S rRNA to m62A2058. This modificationsterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multi-drug resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, while 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 α4, α5 and the α5-α6 linker are essential for methyltransferase function including: an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine, and basic residues in α5 and the α5-α6 linker which 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.

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