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Nucleic Acids Research, Vol 27, Issue 20 4059-4070, Copyright © 1999 by Oxford University Press

ARTICLES

Assignment of the L30-mRNA complex using selective isotopic labeling and RNA mutants

H Mao and JR Williamson
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The helix-loop-helix structure formed in the pre-mRNA and the mRNA of L30, a ribosomal protein from the yeast Saccharomyces cerevisiae, serves as an auto-regulatory binding site for the protein to suppress the L30 synthesis upon overproduction. Using a 33-nucleotide model RNA, the structures of the L30 binding site RNA in the presence and absence of the protein were investigated using nuclear magnetic resonance (NMR) spectroscopy. Homonuclear and(13)C/(15)N-based resonance assignments and spectral comparisons indicated that the purine-rich internal loop is dynamic in the free RNA but becomes ordered in the presence of L30 protein. Although the resonances in the loop region are sharper and more disperse in the bound RNA, their assignment was extremely challenging, due to spectral complexity and broadened resonances caused by local dynamics. Two strategies, namely selective(13)C/(15)N-labeling and NMR analyses of five complexes with RNA mutants, were used to overcome these difficulties. Only using these approaches could assignment of the internal loop resonances and identification of the unusual NOEs and nucleotide conformations within the internal loop be made. In the case of structural determination of the L30-mRNA complex, it was critical to be able to take advantage of the available biochemical information in order to complete the structure determination.
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S. A. WHITE, M. HOEGER, J. J. SCHWEPPE, A. SHILLINGFORD, V. SHIPILOV, and J. ZARUTSKIE
Internal loop mutations in the ribosomal protein L30 binding site of the yeast L30 RNA transcript
RNA, March 1, 2004; 10(3): 369 - 377.
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