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.
    Full-text · Article · Mar 2014 · Protein & Cell
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    • "Several GTPases also facilitate 50S subunit assembly, namely RgbA, YsxC and YphC in B. subtilis and Der and ObgE in E. coli (Hwang & Inouye, 2006; Jiang et al., 2006; Matsuo et al., 2006; Schaefer et al., 2006; Uicker et al., 2006; Wicker-Planquart et al., 2008). In addition, using a proteomic approach, several candidate ribosome-maturation factors have been identified (Jiang et al., 2007). "
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    ABSTRACT: SUMMARY A temperature-sensitive mutation in rplB, designated rplB142, encodes a missense mutation at position 142 (His [CAT] to Leu [CTT]) of ribosomal protein L2. The strain carrying the mutation grew more slowly than the wild type, even at low temperatures, most likely due to the formation of defective 70S ribosomes and the accumulation of incomplete 50S subunits (50S* subunits). Gel analysis indicated that not only the amounts of L2 protein but also the amounts of L16 protein were reduced in ribosomes prepared from the rplB142 mutant 90 min after increasing the growth temperature to 45°C. These results suggest that the assembly of the L16 protein into the 50S subunit requires the native L2 protein. The H142L mutation in the defective L2 protein affected sporulation as well as growth, even at the permissive temperature. A suppressor mutation that restored both growth and sporulation of the rplB142 mutant at low temperature was identified as a single base deletion located immediately upstream of the yaaA gene that resulted in an increase in its transcription. Furthermore, genetic analysis showed that enhanced synthesis of YaaA restores the functionality of L2 (H142L) by facilitating its assembly into 50S subunits.
    Full-text · Article · Mar 2014 · Microbiology
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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.
    Full-text · Article · Oct 2013 · PLoS ONE
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