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

Abstract

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 59 terminus of 16S rRNA, in the 300 region of its 59 domain, and in the 1070 region of its 39-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.

Interdependencies govern multidomain architecture in ribosomal small subunit assembly

“…(D,E) Tethered probing sites on S8. The positions of cysteine substitutes (Lancaster et al 2000) are indicated by number and distinctly colored. D is oriented as in C, E depicting the molecule rotated by 180°along the y-axis.…”

“…However, data from X-ray crystallography (see Fig. 1B; Lancaster et al 2000;Schuwirth et al 2005) as well as solution (see Fig. 1B; Powers and Noller 1995) and directed hydroxyl radical probing (Lancaster et al 2000) expanded our understanding of S8 interaction with 16S rRNA to include a second site at the junction of helices 25, 26, and 26a (see Fig.…”

“…1B). The existence of two separate sites led to speculation that S8 plays a role in compacting the central domain during 59-39 assembly in vivo (Lancaster et al 2000). Moreover, within the tertiary structure of the small subunit, S8 is proximal to the junction of body and platform (see Fig.…”

“…The technique of directed hydroxyl radical probing has many applications, including the localization of binding sites, the determination of orientation of a protein with respect to an RNA or DNA molecule (Culver and Noller 2000a;Lancaster et al 2000;Chen and Hahn 2003;Xu et al 2008), the characterization of the RNA or DNA environment of the Fe(II)-derivatized protein, and monitoring conformational changes or dynamics associated with the incorporation of r-proteins with rRNA Culver 2003, 2004;Dutca and Culver 2008). In fact, a previous study had been carried out using Fe(II)-S8 to localize its binding site in the 70S ribosome (Lancaster et al 2000).…”

“…In fact, a previous study had been carried out using Fe(II)-S8 to localize its binding site in the 70S ribosome (Lancaster et al 2000). These probing experiments revealed that Fe(II)-S8 could cleave elements within the head, body, and platform of the 30S subunit, thereby suggesting that probing mediated by Fe(II)-S8 would allow assembly of these domains to be monitored.…”