Poster abstracts
Poster number 30 submitted by Sourav Kumar Dey
Analysis of the debranching reaction with backbone branched RNA substrates
Sourav Kumar Dey (Department of Chemistry, Carnegie Mellon University.), Linda A. Peteanu (Department of Chemistry, Carnegie Mellon University.), Subha R. Das (Department of Chemistry, Carnegie Mellon University.)
Abstract:
The lariat RNA generated during pre-mRNA splicing includes a branch-point adenosine residue that is linked through the 2'-O position to the 5'-end of the RNA sequence. This 2'-5'-phosphodiester linkage in the lariat RNA backbone is debranched by the lariat debranching enzyme (Dbr1p). Following debranching, some introns can participate in highly important biological processes like snoRNA biogenesis, microRNA pathways etc. In the Das lab we have facile access to 2'-5' phosphodiester linked backbone branched RNA (bbRNA) which are mimics of the lariat introns and that enables us to study the cleavage mechanism of Dbr1p. To examine the kinetics of the debranching reaction, we have synthesized a dual labeled bbRNA with Cy3 and Cy5 dyes that acts as donor and acceptor in a FRET based assay. By following the increase in the donor emission, we have been able to find the kinetics constants of the Dbr1p enzyme. We have also synthesized an analogue of the native substrate where the 2'-5' phosphodiester bond is replaced by a trizole linkage. This 'click-branched' RNA serves as a non-cleavable substrate mimic of Dbr1p. Using the FRET based kinetics we show that this 'click-branched' RNA is a competitive inhibitor of Dbr1p. We examine the dependence of the inhibition activity on the length of the stem and the 2'-5' branch of the 'click-branched' RNA. We are currently using these 'click-branched' RNAs in single molecule FRET studies to understand its structure, dynamics and binding to Dbr1p. An initial design for single molecule FRET studies of the branched RNA and the debranching reaction is also presented.
Keywords: Debranching enzyme , Backbone branched RNA, FRET