Interdependencies govern multidomain architecture in ribosomal small subunit assembly

Abstract

The 30S subunit is composed of four structural domains, the body, platform, head, and penultimate/ultimate stems. The functional integrity of the 30S subunit is dependent upon appropriate assembly and precise orientation of all four domains. We examined 16S rRNA conformational changes during in vitro assembly using directed hydroxyl radical probing mediated by Fe(II)-derivatized ribosomal protein (r-protein) S8. R-protein S8 binds the central domain of 16S rRNA directly and independently and its iron derivatized substituents have been shown to mediate cleavage in three domains of 16S rRNA, thus making it an ideal probe to monitor multidomain orientation during assembly. Cleavages in minimal ribonucleoprotein (RNP) particles formed with Fe(II)-S8 and 16S rRNA alone were compared with that in the context of the fully assembled subunit. The minimal binding site of S8 at helix 21 exists in a structure similar to that observed in the mature subunit, in the absence of other r-proteins. However, the binding site of S8 at the junction of helices 25-26a, which is transcribed after helix 21, is cleaved with differing intensities in the presence and absence of other r-proteins. Also, assembly of the body helps establish an architecture approximating, but perhaps not identical, to the 30S subunit at helix 12 and the 59 terminus. Moreover, the assembly or orientation of the neck is dependent upon assembly of both the head and the body. Thus, a complex interrelationship is observed between assembly events of independent domains and the incorporation of primary binding proteins during 30S subunit formation.

Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly

“…Therefore, it has been hypothesized that r-proteins participate in the dynamic folding and stabilization of rRNA during ribosome assembly and function (Klein et al 2004). This has been supported by thermodynamic and kinetic studies on in vitro reconstitution of bacterial ribosomes (Recht and Williamson 2004;Holmes and Culver 2005;Ramaswamy and Woodson 2009a,b;Woolstenhulme and Hill 2009;Calidas and Culver 2010;Mayerle and Woodson 2013). In eukaryotes, conserved r-proteins have also evolved extra amino acid sequences that are largely found in either carboxy-or amino-terminal domains Yusupova and Yusupov 2014).…”

Ribosome RNA Assembly Intermediates Visualized in Living Cells

“…Significant strides have been made toward understanding ribosome assembly through in vitro experiments, 10–12 and recent work has explored the protein and RNA compositions of assembly intermediates in cells. 2–4 In vitro and in-cell assembly mechanisms differ.…”

The N-terminal extension of S12 influences small ribosomal subunit assembly in Escherichia coli

“…6D). Our previous work suggested that alternative conformations of 16S rRNA existed at nucleotides 566-568 in a minimal complex lacking all 5 ′ domain binding proteins and that the N-terminal segment of S12 played a role in assembly of the interdomain region between the SSU body and platform (Calidas and Culver 2011). In addition to this direct effect, upon deletion of the N-terminus of S12, differences in reactivity have been observed that are distal to S12 in SSUs (see Fig.…”

“…In addition to binding rRNA, the extension also interacts with S8 and S17, and these three r-proteins participate in the assembly of the body, shoulder, and platform of the SSU (see Fig. 1B; Stern et al 1988a,b;Svensson et al 1988;Schuwirth et al 2005;Calidas and Culver 2011). Little is known of the role of the extension in the structure and function of the SSU.…”