Book of Abstracts: Albany 2009

category image Albany 2009
Conversation 16
June 16-20 2009
© Adenine Press (2008)

Splicing Mechanisms: Lessons from Single-Molecule Spectroscopy

Splicing is an essential step in the maturation reaction of eukaryotic pre-mRNA in which intervening sequences (introns) are removed from the coding sequences (exons). The spliceosome is a dynamic assembly of five snRNAs and numerous proteins that catalyzes splicing. U2 and U6 are two snRNAs that form an RNA complex strictly required for both steps of splicing. Major conformational changes are expected to take place during the assembly and catalysis of the spliceosome.

We have developed a single-molecule fluorescence assay to study the structural dynamics of a protein free U2-U6 complex from yeast. Our data clearly show a Mg2+-induced large amplitude conformation change of the U2-U6 complex. In the absence of Mg2+ helix I and the U6-ISL are in close proximity, while in the presence of Mg2+ these two helices are far from each other. This conformational change consists of a two-step process with a previously unobserved obligatory folding intermediate. The first step is Mg2+-dependent, while the second step corresponds to a junction migration that results in the formation the genetically conserved Helix IB. Point mutations in highly conserved regions indicate that the observed dynamics in vitro correlate with spliceosomal activation in vivo. Furthermore, deletion of the highly conserved nucleotide U80, which has been involved in catalysis, shows that this nucleotide plays an important role in stabilizing one of the observed conformations, implicating that this conformation may be important for catalysis.

David Rueda
Zhuojun Guo
Krishanthi Karunatilaka

Department of Chemistry
Wayne State University
Detroit, MI 48202

Email: rueda@chem.wayne.edu