Directed hydroxyl radical probing of 16S ribosomal RNA in 70S ribosomes from internal positions of the RNA

Directed hydroxyl radical probing of 16S ribosomal RNA from Fe(II) tethered to specific sites within the RNA was used to determine RNA-RNA proximities in 70S ribosomes. We have transcribed 16S ribosomal RNA in vitro as two se parate fragments, covalently attached an Fe(II) probe to a 5'-guanosine-alp ha-phosphorothioate at the junction between the two fragments, and reconsti tuted 30S subunits with the two separate pieces of RNA and the small subuni t proteins. Reconstituted 30S subunits capable of association with 50S subu nits were selected by isolation of 70S ribosomes. Hydroxyl radicals, genera ted in situ from the tethered Fe(II), cleaved sites in the 16S rRNA backbon e that were close in three-dimensional space to the Fe(II), and a primer ex tension was used to identify these sites of cleavage. Two sets of 16S ribos omal RNA fragments, 1-360/361-1542 and 1-448/449-1542, were reconstituted i nto active 30S subunits. Fe(II) tethered to position 361 results in cleavag e of 16S rRNA around nucleotides 34, 160, 497, 512, 520, 537, 552, and 615, as well as around positions 1410, 1422, 1480, and 1490. Fe(II) tethered to position 449 induces cleavage around nucleotide 488 and around positions 4 2 and 617. Fe(II) tethered to the 5' end of 16S rRNA induces cleavage of th e rRNA around nucleotides 5, 601, 615, and 642. These results provide const raints for the positioning of these regions of 16S rRNA, for which there ha s previously been only limited structural information, within the 30S subun it.