Poster abstracts
Poster number 107 submitted by Chad Ratterman
Lariat Debranching Enzyme Cleavage of Backbone Branched RNAs With Non-Canonical Branch-Point Residues
Timothy Ratterman (Department of Chemistry, Carnegie Mellon University), Stephanie Mack (Department of Chemistry, Carnegie Mellon University), Subha Das (Department of Chemistry, Carnegie Mellon University)
Abstract:
In splicing, lariat introns are formed typically with a conserved branch-point adenosine. Lariat debranching enzyme (Dbr1p) that specifically cleaves the 2',5'-phosphodiester linkage in lariat RNA thus encounters the branch-point adenosine in natural substrates. Preliminary data from our lab suggests that S. cerevisiae Dbr1p cleaves backbone branched RNAs (bbRNAs) in which the branch-point residue is not adenosine. Recent crystal structures of the active site of Dbr1p from E. histolytica show how the branch-point residue is accommodated in the active site. Here we describe our synthesis of dual fluorescently labeled bbRNAs with C, G and U besides A as branch-point residues. These dual-labeled bbRNAs enable real time kinetics assay of the Dbr1p cleavage reaction. From these assays we analyze the binding and cleavage activity of E. histolytica Dbr1p towards these bbRNAs with non-canonical branch-point residues.
Keywords: Dbr1p, bbRNA