Ribosomal protein S8, which is essential for the
assembly of the central domain of 16S rRNA, is one of the
most thoroughly studied RNA-binding proteins. To map its
surrounding RNA in the ribosome, we carried out directed
hydroxyl radical probing of 16S rRNA using Fe(II) tethered
to nine different positions on the surface of protein S8
in 70S ribosomes. Hydroxyl radical-induced cleavage was
observed near the classical S8-binding site in the 620
stem, and flanking the other S8-footprinted regions of
the central domain at the three-helix junction near position
650 and the 825 and 860 stems. In addition, cleavage near
the 5′ terminus of 16S rRNA, in the 300 region of
its 5′ domain, and in the 1070 region of its 3′-major
domain provide information about the proximity to S8 of
RNA elements not directly involved in its binding. These
data, along with previous footprinting and crosslinking
results, allowed positioning of protein S8 and its surrounding
RNA elements in a 7.8-Å map of the Thermus thermophilus
70S ribosome. The resulting model is in close agreement
with the extensive body of data from previous studies using
protein–protein and protein–RNA crosslinking,
chemical and enzymatic footprinting, and genetics.