The Escherichia coli GTPase CgtAE Is Involved in Late Steps of Large Ribosome Assembly

Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University, Ann Arbor, MI 48109-1048, USA.
Journal of Bacteriology (Impact Factor: 2.81). 11/2006; 188(19):6757-70. DOI: 10.1128/JB.00444-06
Source: PubMed


The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood.
Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not
clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtAE, in late steps of large ribosomal subunit biogenesis. CgtAE belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function.
Mutations in CgtAE cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtAE mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtAE is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover,
we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized
proteins) and define possible temporal relationships between these proteins and CgtAE. We also show that purified CgtAE associates with purified ribosomal particles in the GTP-bound form. Finally, CgtAE cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.

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    • "sucrose cushion (Guo et al., 2013). Notably, a previous study showed that the salt concentration during purification could largely change the protein profile of isolated in vivo 50S assembly intermediates (Jiang et al., 2006). And it is known that the salt concentration is an important factor for reconstitutions of ribosomal subunits in in vitro. "
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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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    • "Gradients were balanced and submitted to ultracentrifugation in an SW50.1 (Beckman) rotor at 41K RPM for 1.5 h at 4°C. After ultracentrifugation, fractionation was performed as previously described [22]. Separation of dissociated subunits was done by ultracentrifugation on 20% sucrose cushions in an SW40TI (Beckman) rotor at 23 K RPM for 15 h at 4°C. "
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    ABSTRACT: The decrease in proline transport by the proline porter ProP in a ΔproQ strain has been well documented; however, the reason for this phenotype remains undefined. Previous studies have speculated that ProQ facilitates translation of proP mRNA. Here, we demonstrate that ProQ is enriched in the polysome fractions of sucrose gradient separations of E. coli lysates and the 30S fractions of lysates separated under conditions causing ribosomal subunit dissociation. Thus, ProQ is a bona fide ribosome associated protein. Analysis of proQ constructs lacking predicted structural domains implicates the N-terminal domain in ribosome association. Association with the ribosome appears to be mediated by an interaction with the mRNA being translated, as limited treatment of lysates with Micrococcal Nuclease maintains ribosome integrity but disrupts ProQ localization with polysomes. ProQ also fails to robustly bind to mRNA-free 70S ribosomes in vitro. Interestingly, deletion of proP does not disrupt the localization of ProQ with translating ribosomes, and deletion of proP in combination with the proU operon has no effect on ProQ localization. We also demonstrate that ProQ is necessary for robust biofilm formation, and this phenotype is independent of ProP. Binding studies were carried out using tryptophan fluorescence and in vitro transcribed proP mRNAs. proP is transcribed from two differentially regulated promoters, and ProQ interacts with proP mRNA transcribed from both promoters, as well as a control mRNA with similar affinities. In total, these data suggest that ProQ is positioned to function as a novel translational regulator, and its cellular role extends beyond its effects on proline uptake by ProP.
    PLoS ONE 10/2013; 8(10):e79656. DOI:10.1371/journal.pone.0079656 · 3.23 Impact Factor
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    • "Our structural data show that L16 is incompatible with H38 in the state II, implying that the binding of L16 would require the breaking of existing non-native interaction between H38 and the L7/L12 stalk base and the reestablishing the native interaction between H38 and the 5S rRNA (Figure 4). Consistently, L16 is exactly a common protein detected to be missing from various forms of premature particles (45–49) (Supplementary Table S3). Along this line, early in vitro reconstitution experiments showed that the incorporation of L16 into the late-stage particles requires a large activation energy and is temperature dependent (50). "
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    ABSTRACT: Ribosome assembly is a process fundamental for all cellular activities. The efficiency and accuracy of the subunit assembly are tightly regulated and closely monitored. In the present work, we characterized, both compositionally and structurally, a set of in vivo 50S subunit precursors (45S), isolated from a mutant bacterial strain. Our qualitative mass spectrometry data indicate that L28, L16, L33, L36 and L35 are dramatically underrepresented in the 45S particles. This protein spectrum shows interesting similarity to many qualitatively analyzed 50S precursors from different genetic background, indicating the presence of global rate-limiting steps in the late-stage assembly of 50S subunit. Our structural data reveal two major intermediate states for the 45S particles. Consistently, both states severally lack those proteins, but they also differ in the stability of the functional centers of the 50S subunit, demonstrating that they are translationally inactive. Detailed analysis indicates that the orientation of H38 accounts for the global conformational differences in these intermediate structures, and suggests that the reorientation of H38 to its native position is rate-limiting during the late-stage assembly. Especially, H38 plays an essential role in stabilizing the central protuberance, through the interaction with the 5S rRNA, and the correctly orientated H38 is likely a prerequisite for further maturation of the 50S subunit.
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