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Book of Abstracts: Albany 2003

category image Albany 2003
Conversation 13
Abstract Book
June 17-21 2003

Dynamics, Base Ionization and Metal Binding in the Structure of U6 RNA

We are investigating the structure and function of spliceosomal RNAs.

The spliceosome is a large ribonucleoprotein particle containing 5 small nuclear RNAs and more than 70 proteins. Biochemical evidence to date suggests that the U2 and U6 spliceosomal RNAs comprise at least part of the catalytic core of the spliceosome, and may directly contribute to the catalysis of pre-messenger RNA splicing. We have determined the NMR structure of the highly conserved U6 intramolecular stem-loop (U6 ISL) RNA, which binds a divalent metal ion that is required for the first step of splicing (1). Metal ion binding is modulated by the protonation state of an adjacent C-A+ wobble base pair. The folding free energy gained by adenine protonation (ΔΔG) is estimated to be -0.7±0.3 kcal/mol. To better understand the structural requirements of the U6 ISL, we have also solved the structure of a lethal point mutation within the yeast U6 ISL (2). We find that the point mutation disrupts the C-A+ wobble pair by forming a non-native Watson-Crick pair, resulting in an overall hyperstabilization of the structure and loss of the ionizing nitrogen. Analysis of relaxation rates provides evidence for dynamic motions within the U6 ISL and suggests that an essential nucleotide undergoes a base-flipping motion on the micro-millisecond timescale.

Nicholas J. Reiter
Dipali G. Sashital
Samuel E. Butcher

University of Wisconsin-Madison
Department of Biochemistry
433 Babcock Dr.
Madison, WI 53706
butcher@nmrfam.wisc.edu

References and Footnotes
  1. Huppler, A., Nikstad, L. J., Allmann, A. M., Brow, D. A. & Butcher, S. E., Nature Struct. Biol. 9, 431-435 (2002).
  2. Sashital, D.G, Allmann, A. M., Van Doren, S. R. & Butcher, S. E. Biochemistry, 42, 1470-1477 (2003).