Probing the phosphates of the Escherichia coli ribosomal 16S RNA in its naked form, in the 30S subunit, and in the 70S ribosome

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Abstract
Ethylnitrosourea is an alkylating reagent which preferentially modifies phosphates in nucleic acids. It was used to map phosphates in naked Escherichia coli 16S rRNA engaged in tertiary interactions through hydrogen bonds or ion coordination. Of the phosphates, 7% are found involved in such interactions, and 57% of them are located in loops or interhelical regions, where they are involved in maintaining local intrinsic structures or long-distance tertiary interactions. The other phosphates (43%) are found in helical regions. These phosphates often occur at the proximity of bulged nucleotides or in irregular helices containing noncanonical base pairs (and bulges) and are assumed to bind cations in order to neutralize negative charges and to stabilize unusual phosphate backbone folding. In the 30S subunit, ENU allowed mapping of phosphates in contact with proteins. The RNA is not uniformly engaged in RNA/protein interactions. Region 1-51, 250-310, 567-612, 650-670, and 1307-1382 are particularly buried whereas the 3''-terminal domain and the 5''-proximal region (nucleotides 53-218) are exposed. The conformation of 16S rRNA is not drastically affected by protein binding, but conformational adjustments are detected in several defined regions. They are found in the 5'' domain (region (147-172), in the central domain (region 827-872), in the 3'' major domain (nucleotides 955-956, 994, 1054, 1181, 1257, and 1262-1263), and in the 3''-terminal domain (around 1400). The 50S subunit shields clusters of phosphates located at the subunit interface. The most extensive protections are observed in the 3''-terminal domain (1490-1542), in the central region of the molecule (770-930), and in the upper 3'' major domain. The binding of the 50S subunit also cause changes of reactivity in the 3'' part of the central domain (around 830), most likely reflecting dynamic properties in this region.