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Ribosomal protein S8 of Escherichia coli plays a key role in 30S ribosomal subunit assembly through its interaction with 16S rRNA. S8 also participates in the translational regulation of ribosomal protein expression through its interaction with spc operon mRNA. The binding site for protein S8 within the 16S rRNA encompasses nucleotides G588 to G604 and C634 to C651 and is composed of two base paired helical regions that f lank a phylogenetically conserved core element containing nine residues. We have investigated the structure of the rRNA binding site for S8 both in the free state and in the presence of protein using NMR spectroscopy. The integrity of the two helical segments has been verified, and the presence of G597⅐C643 and A596⅐U644 base pairs within the conserved core, predicted from comparative analysis, have been confirmed. In addition, we have identified a base triple within the core that is composed of residues A595⅐(A596⅐ U644). The NMR data suggest that S8-RNA interaction is accomplished without significant changes in the RNA. Nonetheless, S8 binding promotes formation of the U598⅐A640 base pair and appears to stabilize the G597⅐C643 and A596⅐U644 base pairs.The structural features of RNA that are important for RNAspecific protein recognition have only recently come under investigation using solution state methods (1-3). The assembly and maturation of ribosomes are critically dependent upon a large network of protein-RNA interactions, and a detailed description of the underlying structures is essential for understanding the biological activity of these particles in protein synthesis (4, 5). The binding of ribosomal protein S8 within the central domain of the 16S rRNA constitutes one of the first steps in 30S subunit assembly in Escherichia coli (6). In addition, the specific interaction of protein S8 with spc operon mRNA mediates translational regulation of the expression of S8 and a number of other ribosomal proteins (7). The association of S8 with its rRNA and mRNA binding sites has been extensively characterized by nuclease protection (8, 9), comparative sequence analysis (9), chemical modification (10-13), cross-linking (14), and site-directed mutagenesis (9,11,15). While these investigations have provided important information about the elements of RNA primary and secondary structure that are involved in S8-RNA interaction, we present here the first three-dimensional (3D) structure of the binding site for protein S8 within the 16S rRNA determined using NMR techniques.The binding site for protein S8 is located within helix 21 of the 16S rRNA (Fig. 1A) and comprises two helical segments interrupted by a core element of irregular structure that spans residues 595-598 and 640-644 (Fig. 1B). The nucleotides of the core element are very highly conserved among prokaryotic 16S rRNAs (16), and nearly all of them have been shown to be crucial for recognition by protein S8 through specific base substitutions (9, 11, 13). Significantly, the regulatory binding site for protein S8 in spc mRNA can ado...

An RNA oligonucleotide that contains the binding site for Escherichia coli ribosomal protein S8 was prepared with uniform 15N isotopic enrichment and uniform deuterium enrichment at all non-exchangeable sites using enzymatic methods. The RNA binding site, which contains 44 nt, forms a hairpin in solution and requires Mg2+for proper folding. The longitudinal magnetization recovery rates of the exchangeable protons were compared for the [2H,15N]-enriched RNA molecule and for the corresponding fully [2H,15N]-enriched RNA hairpin. It was found that 1H-1H dipolar relaxation significantly contributes to the recovery of exchangeable proton longitudinal magnetization. The exchangeable proton resonance line widths were less affected by deuteration, indicating that chemical exchange with H2O remains the dominant mechanism of transverse magnetization relaxation. Nevertheless, deuteration of this RNA hairpin was found to enhance the sensitivity of NOE-based experiments relative to the fully protonated hairpin and to simplify 2D NMR spectra. The increased signal-to-noise ratio facilitated the assignment of the cytidine amino resonances and several of the purine nucleotide amino resonances and permitted the identification of NOE crosspeaks that could not be observed in spectra of the fully protonated RNA hairpin.

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