Site-directed hydroxyl radical probing of 30S ribosomal subunits by using Fe(II) tethered to an interruption in the 16S rRNA chain

Two irt vitro transcripts, one corresponding to the 5' and central domains (residues 1-920) of 16S rRNA and the other corresponding to its 3' domain ( residues 922-1542), assemble efficiently in trans with 30S ribosomal protei ns to form a compact ribonucleoprotein particle that cosediments with natur al 30S subunits. Isolated particles are similar in appearance to natural 30 S subunits with electron microscopy and contain a full complement of the sm all subunit ribosomal proteins. The particles have a reduced ability to bin d tRNA (attributable to the location of the discontinuity in a conserved re gion of the rRNA) near features that have been implicated in tRNA binding. Association of these two halves of 16S rRNA in trans must be stabilized by either previously unidentified RNA-RNA contacts or interactions mediated by ribosomal proteins because there are no known direct interactions between them. The trans construct was used to probe the three-dimensional RNA neigh borhood around position 922 of 16S rRNA by generating hydroxyl radicals fro m Fe(II) tethered to the 5' end of the 3' transcript. Hydroxyl radical-indu ced cuts in the 16S rRNA chain were localized by primer extension to nucleo tides 923-929 and 1192-1198, providing evidence for the mutual proximity of the 920 and 1192 regions.