Directed hydroxyl radical probing of 16S rRNA in the ribosome: Spatial proximity of RNA elements of the 3′ and 5′ domains

Abstract

We have shown previously that directed hydroxyl radical probing of 16S rRNA from Fe(II) tethered to specific sites within the RNA gives valuable information about RNA–RNA proximities in 70S ribosomes. Here, we extend that study and present probing data from nt 424 in 16S rRNA. To tether an Fe(II) to position 424 in the rRNA we created a specific discontinuity in the RNA by in vitro transcription of the RNA as two separate fragments corresponding to nt 1–423 and 424–1542. An Fe(II)-BABE was covalently attached to a 5′-guanosine-α-phosphorothioate at position 424 and 30S subunits were reconstituted from the two pieces of rRNA and the small subunit proteins. Reconstituted 30S subunits capable of associating with 50S subunits were selected by isolation of 70S ribosomes. Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved positions in the RNA backbone that were close in three-dimensional space to the Fe(II), and the sites of cleavage were identified using primer extension. Fe(II) tethered to position 424 induces cleavage around nt 424, 513, and 531 in the 5′-domain of 16S rRNA and around nt 1008, 1029, 1044, and 1208 in the 3′-domain of 16S ribosomal RNA. These data constrain the positions of the 420, 1015, 1030 and 1000/1040 helices, for which there is little structural information. Since the 5′- and 3′-domains of 16S rRNA constitute the body and head, respectively, of 30S subunits, these findings provide direct evidence for proximity of RNA elements in the body and head of 30S.