Interdependencies govern multidomain architecture in ribosomal small subunit assembly

Department of Biology, University of Rochester, Rochester, New York 14627, USA.
RNA (Impact Factor: 4.94). 02/2011; 17(2):263-77. DOI: 10.1261/rna.2332511
Source: PubMed


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 5' 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.

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    • "Nevertheless, the in vitro reconstitution of ribosomal subunit free of assembly factors has remained to be a classical biochemical system for decades, with a focus on studying the binding interdependence among ribosomal proteins [for examples, see (Grondek and Culver, 2004; Mizushima and Nomura, 1970; Rohl and Nierhaus, 1982)] and metastable rRNA conformational transitions [for examples, see (Calidas and Culver, 2011; Holmes and Culver, 2004, 2005; Powers et al., 1993; Ramaswamy and Woodson, 2009; Stern et al., 1989)] during the assembly process. More recently, time-resolved techniques from pulse-labeling based quantitative mass-spectrometry (QMS) (Mulder et al., 2010; Talkington et al., 2005) and synchrotron X-ray footprinting (Adilakshmi et al., 2008) have further generated a large amount of real-time data for various protein binding and rRNA folding events in the process of the in vitro 30S subunit assembly. "
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    ABSTRACT: The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3′ domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5′ end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation. Electronic supplementary material The online version of this article (doi:10.1007/s13238-014-0044-1) contains supplementary material, which is available to authorized users.
    Protein & Cell 03/2014; 5(5). DOI:10.1007/s13238-014-0044-1 · 3.25 Impact Factor
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    04/2011, Degree: PhD, Supervisor: Jaanus Remme, Tanel Tenson
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    ABSTRACT: The assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. The ribosomal protein S8 from Aquifex aeolicus (AS8) is unique in that there is a 41-residue insertion in the consensus S8 sequence. In addition, AS8 exhibits an unusually high affinity for the 16S ribosomal RNA, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than the equivalent interaction from Escherichia coli. Deletion analysis demonstrated that binding to the minimal site on helix 21 occurred at the same nanomolar affinity found for other bacterial species. The additional affinity required the presence of a three-helix junction between helices 20, 21, and 22. The crystal structure of AS8 was solved, revealing the helix-loop-helix geometry of the unique AS8 insertion region, while the core of the molecule is conserved with known S8 structures. The AS8 structure was modeled onto the structure of the 30S ribosomal subunit from E. coli, suggesting the possibility that the unique subdomain provides additional backbone and side-chain contacts between the protein and an unpaired base within the three-way junction of helices 20, 21, and 22. Point mutations in the protein insertion subdomain resulted in a significantly reduced RNA binding affinity with respect to wild-type AS8. These results indicate that the AS8-specific subdomain provides additional interactions with the three-way junction that contribute to the extremely tight binding to ribosomal RNA.
    Journal of Molecular Biology 11/2011; 415(3):489-502. DOI:10.1016/j.jmb.2011.10.046 · 4.33 Impact Factor
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