DIRECTED HYDROXYL RADICAL PROBING OF THE RIBOSOMAL-RNA NEIGHBORHOOD OF RIBOSOMAL-PROTEIN S13 USING TETHERED FE(II)

Directed hydroxyl radical probing was used to probe the rRNA neighborh ood around protein S13 in the 30S ribosomal subunit. The unique cystei ne at position 84 of S13 served as a tethering site for attachment of Fe(II)-1-(p-bromoacetamidobenzyl)-EDTA. Derivatized S13 (Fe-C84-S13) w as then assembled into 30S ribosomal subunits by in vitro reconstituti on with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe(II) resulted in cleav age of the RNA backbone in two localized regions of the 3' major domai n of 16S rRNA. One region spans nt 1308-1333 and is close to a site pr eviously crosslinked to S13. A second set of cleavages is found in the 950/1230 helix. Both regions have been implicated in binding of S13 b y previous chemical footprinting studies using base-specific chemical probes and solution-based hydroxyl radical probing. These results plac e both regions of 16S rRNA in proximity to position C84 of S13 in the three-dimensional structure of the 30S ribosomal subunit.