Assembly of the 5′ and 3′ Minor Domains of 16S Ribosomal RNA as Monitored by Tethered Probing from Ribosomal Protein S20

Abstract

The ribosomal protein (r-protein) S20 is a primary binding protein. As such, it interacts directly and independently with the 5' domain as well as the 3' minor domain of 16S ribosomal RNA (rRNA) in minimal particles and the fully assembled 30S subunit. The interactions observed between r-protein S20 and the 5' domain of 16S rRNA are quite extensive, while those between r-protein S20 and the 3' minor domain are significantly more limited. In this study, directed hydroxyl radical probing mediated by Fe(II)-derivatized S20 proteins was used to monitor the folding of 16S rRNA during r-protein association and 30S subunit assembly. An analysis of the cleavage patterns in the minimal complexes [16S rRNA and Fe(II)-S20] and the fully assembled 30S subunit containing the same Fe(II)-derivatized proteins shows intriguing similarities and differences. These results suggest that the two domains, 5' and 3' minor, are organized relative to S20 at different stages of assembly. The 5' domain acquires, in a less complex ribonucleoprotein particle than the 3' minor domain, the same architecture as observed in mature subunits. These results are similar to what would be predicted of subunit assembly by the 5'-to-3' direction assembly model.

Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5′-domain assembly

“…Protein S17 interacts with 16S helix 11 and also indirectly stabilizes tertiary interactions in the lower half of the 5 ′ domain (Ramaswamy and Woodson 2009a). Binding of protein S20 to helices 6, 8, 9, and 10 stabilizes those helix junctions and mediates interactions with helix 44 later in 30S assembly (Dutca and Culver 2008;Ramaswamy and Woodson 2009a).…”

Comprehending a Molecular Conundrum: Functional Studies of Ribosomal Protein Mutants from Salmonella enterica Serovar Typhimurium

“…The protein (Figure 10) is located at the base of the body of the 30S subunit, distal to the mRNA binding channel and subunit interface. However S20 has been shown to interact with helix 44 of the 16S rRNA during 30S assembly and in mature 30S particles (Brodersen et al, 2002;Dutca & Culver, 2008). High-resolution crystal structures have shown that this helix stretches across the entire length of the 30S body and forms part of the A-and P-sites where mRNA binding occurs (Schluenzen et al, 2000) and it also forms many bridging contacts with the 50S subunit (Selmer et al, 2006).…”

Interdependencies govern multidomain architecture in ribosomal small subunit assembly

“…Previous studies have demonstrated that differences in cleavage at specific sites between two Fe(II)-derivatized complexes assembled and probed simultaneously can be interpreted as indicating alternative conformations or dynamics of 16S rRNA in those complexes (Jagannathan and Culver 2004;Dutca and Culver 2008). Therefore, Fe(II)-S8 containing RNPs of different complexities were probed to dissect assembly events that occur subsequent to S8 association with 16S rRNA and to reprise patterns of Fe(II)-S8 cleavage observed in 30S subunits.…”

“…In order to form the RNPs, the reactions were incubated at 42°C for 20 min. These were then cooled on ice for 10 min and purified by passing them through spin columns with a speed of 2500 rpm for 3.5 min as described (Dutca and Culver 2008). In order to minimize non-directed probing, 1.6 mL of 125 mM EDTA was added to yield a final concentration of 2 mM EDTA per reaction except to the complexes depicted in Figure 2C.…”

“…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).…”