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Access Type

WSU Access

Date of Award

1-1-2010

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

David Rueda

Abstract

Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging

from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5

and U6) involved in substrate positioning and catalysis. It has been postulated, but never

shown experimentally, that the U2-U6 complex adopts at least two conformations that

reflect different activation states. We have used single-molecule fluorescence to probe the

structural dynamics of a protein-free RNA complex modeling U2-U6 from yeast and

mutants of highly conserved regions of U2-U6. Our data show the presence of at least

three distinct conformations in equilibrium. The minimal folding pathway consists of a

two-step process with an obligatory intermediate. The first step is strongly magnesium

dependent, and we provide evidence suggesting that the second step corresponds to the

formation of the genetically conserved helix IB. Site-specific mutations in the highly

conserved AGC triad and the U80 base in U6 suggest that the observed conformational

dynamics correlate with residues that have an important role in splicing. We also report

the first direct structural evidence that supports the existence of the base triples in the spliceosomal snRNA U2/U6. These interactions were proposed according to a

corresponding set of base triple interactions discovered in the recent published crystal

structure of the self-splicing group II intron.19-16, 24b-17, 48 We proposed that these base

triples existing in the spliceosomal RNA U2/U6 complex are in the same family of the

ones found in crystal structure of the self-splicing group II intron given the extensive

similarities between the spliceosome and the group II intron. Our data agree very well

with the hypothesis. There are a large number of proteins in the spliceosome that play

vital structural and catalytic roles3,36. RNA Chaperones, like Prp24, help the structural

rearrangements of spliceosomal snRNAs. We report single molecule FRET data showing

that spliceosomal protein Prp24 can induce a conformational change in U2/U6 complex.

This RNA:protein interaction is Mg2+ and protein concentration dependent and inhibits

the binding of Mg2+ to U2/U6.