Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20

The 16S ribosomal RNA neighborhood of ribosomal protein S20 has been mapped , in both 30S subunits and 70S ribosomes, using directed hydroxyl radical p robing. Cysteine residues were introduced at amino acid positions 14, 23, 4 9, and 57 of S20, and used for tethering 1-(p-bromoacetamidobenzyl)-Fe(ll)- EDTA. In vitro reconstitution using Fe(ll)-derivatized S20, together with t he remaining small subunit ribosomal proteins and S63 ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes conta ining Fe(ll)-S20 were purified and hydroxyl radicals were generated from th e tethered Fe(ll). Hydroxyl radical cleavage of the 16S rRNA backbone was m onitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(ll) tethered to each of the four positions, and these patt erns were not significantly different in 30S and 70S ribosomes. Cleavage si tes were mapped to positions 160-200, 320, and 340-350 in the 5' domain, an d to positions 1427-1430 and 1439-1458 in the distal end of the penultimate stem of 16S rRNA, placing these regions near each other in three dimension s. These results are consistent with previous footprinting data that locali zed 320 near these 16S rRNA elements, providing evidence that S20, like S17 , is located near the bottom of the 30S subunit.