The location of protein S8 and surrounding elements of 16S rRNA in the 70Sribosome from combined use of directed hydroxyl radical probing and X-ray crystallography

Ribosomal protein S8, which is essential for the assembly of the central do main of 16S rRNA, is one of the most thoroughly studied RNA-binding protein s. To map its surrounding RNA in the ribosome, we carried out directed hydr oxyl radical probing of 16S rRNA using Fe(II) tethered to nine different po sitions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-ind uced cleavage was observed near the classical S8-binding site in the 620 st em, 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 ad dition, 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 informati on about the proximity to S8 of RNA elements not directly involved in its b inding. These data, along with previous footprinting and crosslinking resul ts, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-Angstrom map of the Thermus thermophilus 70S ribosome. The resulting m odel is in close agreement with the extensive body of data from previous st udies using protein-protein and protein-RNA crosslinking, chemical and enzy matic footprinting, and genetics.