The 16S ribosomal RNA neighborhood of ribosomal
protein S20 has been mapped, in both 30S subunits and 70S
ribosomes, using directed hydroxyl radical probing. Cysteine
residues were introduced at amino acid positions 14, 23,
49, and 57 of S20, and used for tethering
In vitro reconstitution using Fe(II)-derivatized S20, together
with the remaining small subunit ribosomal proteins and
16S ribosomal RNA (rRNA), yielded functional 30S subunits.
Both 30S subunits and 70S ribosomes containing Fe(II)-S20
were purified and hydroxyl radicals were generated from
the tethered Fe(II). Hydroxyl radical cleavage of the 16S
rRNA backbone was monitored by primer extension. Different
cleavage patterns in 16S rRNA were observed from Fe(II)
tethered to each of the four positions, and these patterns
were not significantly different in 30S and 70S ribosomes.
Cleavage sites were mapped to positions 160–200,
320, and 340–350 in the 5′ domain, and 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 dimensions. These results are
consistent with previous footprinting data that localized
S20 near these 16S rRNA elements, providing evidence that
S20, like S17, is located near the bottom of the 30S subunit.