Studies of the Interaction of
            <i>Escherichia coli</i>
            YjeQ with the Ribosome In Vitro

Daigle, Denis M.; Brown, Eric D.

doi:10.1128/jb.186.5.1381-1387.2004

Cited by 99 publications

(187 citation statements)

References 15 publications

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“…2B). Previous work on RsgA from E. coli reported that the activity of the protein is increased in the presence of ribosomes (36,51). To determine the effect of ribosomes on the activity of all four GTPases, 70S ribosomes were purified from the community-acquired methicillin-resistant S. aureus (CA-MRSA) strain LAC* and included in the GTP hydrolysis assays.…”

Section: Resultsmentioning

confidence: 99%

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

Corrigan

Bellows

Wood

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

188

227

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The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Grampositive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Grampositive bacterium Staphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases-RsgA, RbgA, Era, HflX, and ObgE-as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.ribosome | stringent response | tolerance | ppGpp | Staphylococcus aureus

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Section: Resultsmentioning

confidence: 99%

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

Corrigan

Bellows

Wood

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

188

227

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“…Recent studies show that the GTPase activity of E. coli YjeQ, which associates with 30 S subunit, is enhanced in the presence of this subunit in vitro (25,26). Because YlqF bound to the free 50 S subunit, but not 30 S subunit, we next examined the activation of GTPase activity of YlqF by the 50 S subunit.…”

Section: Gtpase Activity Of Ylqf Is Stimulated By the 50 S Ribosomal mentioning

confidence: 99%

“…Another essential bacterial small GTP-binding protein, YjeQ, associates with the 30 S subunit in E. coli. Its GTPase activity is specifically enhanced in the presence of 30 S subunit (25,26). However, the precise actions of GTP-binding proteins on the ribosome remain to be clarified.…”

mentioning

confidence: 99%

The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

Matsuo

Morimoto²,

Kuwano

et al. 2006

Journal of Biological Chemistry

111

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Bacillus subtilis YlqF belongs to the Era/Obg subfamily of small GTP-binding proteins and is essential for bacterial growth. Here we report that YlqF participates in the late step of 50 S ribosomal subunit assembly. YlqF was co-fractionated with the 50 S subunit, depending on the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50 S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF binds to a unique position in 23 S rRNA. Yeast two-hybrid data revealed interactions between YlqF and the B. subtilis L25 protein (Ctc). The interaction was confirmed by the pull-down assay of the purified proteins. Specifically, YlqF is positioned around the A-site and P-site on the 50 S subunit. Proteome analysis of the abnormal 50 S subunits that accumulated in YlqF-depleted cells showed that L16 and L27 proteins, located near the YlqF-binding domain, are missing. Our results collectively indicate that YlqF will organize the late step of 50 S ribosomal subunit assembly.

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“…A linear relationship was observed between the concentration of NS3-MBP and the amount of GTP hydrolyzed within 1 hr at 37° (Figure 3B), while the negative-control MBP protein was incapable of GTP hydrolysis. NS3's intrinsic GTP hydrolysis rate, K cat 4.27 6 0.59 min 21 , is on par with that of heterotrimeric G-proteins (K cat 3-5 min 21 ) (Kaziro et al 1991), although relatively high compared to some circularly permuted GTPases (K cat 0.2 min 21 ) (Daigle and Brown 2004;Himeno et al 2004;Bassler et al 2006;Cladiere et al 2006) and canonical Ras and Rab GTPase members (K cat ,0.01 min 21 ) (Kaziro et al 1991). It is unknown whether GTPase activities of Lsg1/NS3 orthologs can be stimulated by GTPase-activating proteins (GAPs) or other factors that can increase catalytic rate .100-fold (Kaziro et al 1991;Bernards and Settleman 2004).…”

Section: Ns3 Is a Functioning Gtpase And Inactivation Of This Domain mentioning

confidence: 97%

“…It is unknown whether GTPase activities of Lsg1/NS3 orthologs can be stimulated by GTPase-activating proteins (GAPs) or other factors that can increase catalytic rate .100-fold (Kaziro et al 1991;Bernards and Settleman 2004). For instance, the K cat of E. coli YRG member YjeQ is increased 500-fold in the presence of small ribosomal subunits (Daigle and Brown 2004).…”

Section: Ns3 Is a Functioning Gtpase And Inactivation Of This Domain mentioning

confidence: 99%

Regulation of Ribosome Biogenesis by Nucleostemin 3 Promotes Local and Systemic Growth in Drosophila

Hartl

Cao

et al. 2013

Genetics

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Nucleostemin 3 (NS3) is an evolutionarily conserved protein with profound roles in cell growth and viability. Here we analyze cell-autonomous and non-cell-autonomous growth control roles of NS3 in Drosophila and demonstrate its GTPase activity using genetic and biochemical assays. Two null alleles of ns3, and RNAi, demonstrate the necessity of NS3 for cell autonomous growth. A hypomorphic allele highlights the hypersensitivity of neurons to lowered NS3 function. We propose that NS3 is the functional ortholog of yeast and human Lsg1, which promotes release of the nuclear export adapter from the large ribosomal subunit. Release of the adapter and its recycling to the nucleus are essential for sustained production of ribosomes. The ribosome biogenesis role of NS3 is essential for proper rates of translation in all tissues and is necessary for functions of growth-promoting neurons.T HE rate and fidelity of protein synthesis in a cell depend upon the proper assembly of the ribosome. Ribosome biogenesis begins in the nucleolus with the synthesis of ribosomal RNA (rRNA) and culminates in the creation of a fully functional ribosome competent to initiate protein synthesis in the cytoplasm. This process is impressively intricate, requiring 200 factors acting at various levels. Major ribosome biogenesis steps include directing rRNA post-transcriptional modification, 90S cleavage, folding/ assembly of the small (40S) and large (60S) ribosomal subunits, and proper transport from the nucleus to cytoplasm where final maturation occurs (Zemp and Kutay 2007;Henras et al. 2008;Staley and Woolford 2009;Strunk and Karbstein 2009;Panse and Johnson 2010).Export of pre-ribosomal subunits from the nucleus to the cytoplasm requires movement through the nuclear pore complex (Hurt et al. 1999;Moy and Silver 1999; StageZimmermann et al. 2000;Seiser et al. 2006). The pre-60S subunit achieves nuclear export by recruiting nuclear export factors, including the nuclear export signal (NES)-bearing protein Nmd3 (Ho et al. 2000;Gadal et al. 2001). Equally essential is recycling of Nmd3 from the cytoplasm back to the nucleus for subsequent rounds of pre-60S export. The release of Nmd3 appears to be the last step of a highly ordered pathway of ribosome maturation in the cytoplasm (Lo et al. 2010). Studies in Saccharomyces cerevisiae implicated RpL10p and Lsg1p as factors critical for Nmd3 recycling. Loss of either RpL10p or Lsg1p caused accumulation of Nmd3 in the cytoplasm, which precluded its ability to return to the nucleus for additional rounds of pre-60S export (Hedges et al. 2005). Thus, the loss of Nmd3p, RpL10p, or Lsg1p through mutation causes 60S subunit deficiency in the cytoplasm.In Drosophila, a well-known class of mutations, called Minutes affects ribosome assembly. Minutes have delayed development and small or "minute" bristles; mutations affecting 66 of the 88 predicted ribosomal proteins have this phenotype (Marygold et al. 2007 founding member of what came to be a large class of genes that are often haplo-insufficient. Lat...

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Studies of the Interaction of Escherichia coli YjeQ with the Ribosome In Vitro

Abstract: Escherichia coli

Cited by 99 publications

References 15 publications

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

Regulation of Ribosome Biogenesis by Nucleostemin 3 Promotes Local and Systemic Growth in Drosophila

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