A "bulged" double helix in a RNA-protein contact site.

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Abstract
The binding of ribosomal protein L18 affects specific nucleotides in Escherichia coli 5S RNA as detected by dimethyl sulfate alkylation and RNase A digestion of the 5S-L18 complex. Most of the affected nucleotides are clustered and localize a site of RNA-protein interaction in and around the defined central helix of 5S RNA. Chemical carbethoxylation of the native 5S RNA with diethyl pyrocarbonate shows that a striking feature of this region is an unstacked adenosine residue at position 66. This residue exists as a singly bulged nucleotide extending the Fox and Woese centrl helix by 2 base pairs in the E. coli sequence (to positions 16-23/60-68) and in each of 61 (prokaryotic and eukaryotic) aligned 5S RNA sequences. In each case, the singly bulged nucleotide is at the relative position of adenosine-66 in the RNA sequences. The presence of this putative bulged nucleotide appears to have been conserved in 5S RNA sequences throughout evolution, and its identity varies with major phylogenetic divisions. This residue is likely involved in specific 5S RNA-protein recognition or interaction in prokaryotic and eukaryotic ribosomes. The uridine-65 to adenosine-66 internucleotide bond is protected from RNase A digestion in the complex, and carbethoxylation of E. coli adenosine-66 prior to L18 binding affects formation of a stable RNA-protein complex. A region of E. coli 5S RNA protected by the ribosomal protein L18 was thus identified. This region may contain a bulged nucleotide residue important in stable formation of this RNA-protein complex. This bulged residue appears to be evolutionarily conserved and phylogenetically defined in 5S RNA sequences in general, and consideration of other known RNA-protein binding sites shows that such a bulged helix may be a common feature of RNA-protein contact sites.