Structural and functional characterization of an ATP-dependent RNA ligase from Methanobacterium thermoautotrophicum
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The ATP-dependent RNA ligases are members of the covalent nucleotidyltransferase superfamily, which catalyzes the formation of phosphodiester bonds between the 5'-phosphate and 3'-hydroxyl termini of RNA. Several achaeal species encode an ATP-dependent RNA ligase. The Methanobacterium thermoautotrophicum RNA ligase (MthRnl) is a thermophilic ligase that reacts with ATP to form ligase-adenylate, transfers the AMP to the 5'-PO4 of single-stranded RNA to form an RNA-adenylate and catalyzes the intramolecular joining of the two ends to form a covalently closed circular RNA molecule. MthRnl is unique among the polynucleotide ligases in that it forms a homodimeric structure. The C-terminal domain of MthRnl is required for thermoreactivity and strand-joining activity. In this thesis, structural-based mutational analysis is performed on MthRnl to determine how different functional groups come into play during the ligase-adenylation (step 1) versus the subsequent steps of RNA-adenylation (step 2) and phosphodiester formation (step 3), and how the homodimeric quaternary structure contributes to the ligation reaction. Enzyme bearing's alanine substitutions at Lys-97, Asn-99, Asn-102, Arg-104, Thr-117, Arg-118, Glu-151, Tyr-159, Phe-175, Glu-231, Lys-246, Phe-281, Glu-285 and Arg-363 reduce the rate of ligation by at least an order of magnitude. Asn-102 (interacting with ribose 2'-O and 3'-O of AMP), Glu-151 (interacting with ribose 2'-O of AMP), Tyr-159, Phe-175 (aromatic stacking on the adenine ring on the AMP) and Glu-231 (interacting with the metal) are essential in both step 1 and step 3 reactions. Lys-97 (site for covalent AMP attachment) and Lys-246 (interacting with α-phosphate on ATP) are required in step 1 but are dispensable for the isolated sealing step. Asn-99, Arg-104, Thr-117, Arg-118, Phe-281 and Arg-363 are specifically involved in step 3. A putative RNA binding surface on the MthRnl is identified (Tyr-159, Phe-281 and Glu-285) by mutating the residues conserved among the archaeal RNA ligases that are located on the surface of the protein. Furthermore, deletion analysis suggests that fifteen C-terminal amino acids are essential for the maintenance of the quaternary structure and for the ligation activity. Mutational analysis shows that residues Phe-272, Phe-273, Ile-304 and Ile-305, that lie within the dimerization interface, contribute to the formation of a functional dimer and that homodimerization is required for MthRnl's ligation activity.