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.
