Poster abstracts
Poster number 60 submitted by Chad Ratterman
Investigating active site mutants of lariat debranching enzyme with backbone branched RNA
Timothy Chad Ratterman (Chemistry, Carnegie Mellon University), Brian Graham (Biological Sciences, University of Pittsburgh), Grace Wang (Biological Sciences, University of Pittsburgh), Andrew VanDemark (Biological Sciences, University of Pittsburgh), Subha R. Das (Chemistry, Carnegie Mellon University)
Abstract:
Lariat debranching enzyme (Dbr1p) belongs to the binuclear metallophosphoesterase (MPE) superfamily and processes lariat introns formed during RNA splicing. In the splicing reactions, the 2ʹ,5ʹ-phosphodiester bond formed in the lariat intron is specifically cleaved while the 3ʹ,5ʹ-phosphodiester bonds remain intact. Sequence homology studies have identified a high level of conservation throughout the metallophosphoesterase (MPE) domain. Mutational studies have demonstrated that mutation of certain residues within this highly conserved region greatly diminishes or abolishes enzymatic activity in yeast and human Dbr1p. Recent crystal structures of the E. histolytica Dbr1p suggest critical amino acid residues as contacts with the metal centers or branchpoint adenosine. Here we investigate four mutants of the E. histolytica Dbr1p enzyme and assess their ability to cleave a backbone branched RNA substrate compared to the wild type enzyme. In these mutant enzymes, the changes are to active site residues that are putative ligands for the metal ions or branchpoint adenosine binding. Our data suggest that the E. histolytica Dbr1p tolerates mutations to residues suggested to contact the branchpoint of the branched RNA.
Keywords: Dbr1p, E histolytica, Active site mutants