STRUCTURE OF RIBOSOMAL S 15 PROTEIN BINDING SITE RNA

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A minimal binding site in 16S RNA for the S 15 protein from B. stearothermophilus has been identified, which is a 65 nucleotide RNA composed of three helices that meet at a three-way junction. The 88 residue protein forms a well-behaved complex at I mM concentration, and the optimal temperature for well-resolved spectra is 45-C. We have the capability of labeling this large complex with 13C, 15N, and 2H in a variety of different ways. We have prepared four samples 13C-labeled RNA where only one type of nucleotide is labeled with 13C in each sample. In addition, 15N labeled RNA has been prepared, and a complex with 15N-labeled protein has been made. We are in the process of recording survey spectra to obtain initial assignments for this challenging complex. We have prepared a variety of specifically deuterated RNAs: 3',4',5',5" -d4-RNA, and RNA with specific base deuteration. The spectra of the complex are greatly simplified, and we believe that we should be able to obtain most of the sequential assignments of the base, H V and HT protons of the RNA in the complex using homonuclear spectra at 750 MHz.