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

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 a uto-regulatory binding site for the protein to suppress the L30 synthesis u pon overproduction, Using a 33-nucleotide model RNA, the structures of the L30 binding site RNA in the presence and absence of the protein were invest igated using nuclear magnetic resonance (NMR) spectroscopy. Homonuclear and C-13/N-15-based resonance assignments and spectral comparisons indicated t hat the purine-rich internal loop is dynamic in the free RNA but becomes or dered in the presence of L30 protein. Although the resonances in the loop r egion 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 C-13/N-15-labeli ng and NMR analyses of five complexes with RNA mutants, were used to overco me these difficulties, Only using these approaches could assignment of the internal loop resonances and identification of the unusual NOEs and nucleot ide conformations within the internal loop be made, In the case of structur al determination of the L30-mRNA complex, it was critical to be able to tak e advantage of the available biochemical information in order to complete t he structure determination.