Binding of Thiostrepton to a Complex of 23‐S rRNA with Ribosomal Protein L11

Thompson, Jessica; Cundliffe, Eric; Stark, Michael J. R.

doi:10.1111/j.1432-1033.1979.tb13184.x

Cited by 91 publications

(72 citation statements)

References 10 publications

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“…From this result, Egebjerg et al (1990) proposed a model for the tertiary configuration of the L11-thiostrepton binding region in which the two loops protected by thiostrepton lie in close proximity. L11 and thiostrepton are presumed to stabilize such an RNA tertiary structure (Thompson et al, 1979;Draper et al, 1995;Xing and Draper, 1995). This is comparable with the fact that rat L12 and anti-28 S recognize a similar tertiary structure of the eukaryotic GTPase domain.…”

Section: Conserved Protein Binding Features Of the Gtpase Domain-supporting

confidence: 59%

Binding of Mammalian Ribosomal Protein Complex P0·P1·P2 and Protein L12 to the GTPase-associated Domain of 28 S Ribosomal RNA and Effect on the Accessibility to Anti-28 S RNA Autoantibody

Uchiumi

Kominami

1997

Journal of Biological Chemistry

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We have investigated binding of rat ribosomal proteins to the "GTPase domain" of 28 S rRNA and its effect on accessibility to the anti-28 S autoantibody, which recognizes a unique tertiary structure of this RNA domain. Ribosomal protein L12 and P protein complex (P complex) consisting of P0, P1, and P2 both bound to the GTPase domain of rat 28 S rRNA in a buffer containing Mg 2 . Chemical footprinting analysis of their binding sites revealed that the P complex mainly protected a conserved internal loop region comprising residues 1855-1861 and 1920 -1922, whereas L12 protected an adjacent helix region encompassing residues 1867-1878 and 1887-1899. These sites are close to but distinct from the binding site for anti-28 S antibody determined previously. The bindings of P complex and L12 increased the anti-28 S accessibility, as revealed by gel retardation and quantitative immunoprecipitation analyses. In a Mg 2؉ -eliminated condition, the RNA failed to bind to either anti-28 S or L12 but assembled into a complex under their coexistence. However, the RNA retained a property of binding to the P complex even in the absence of Mg 2؉ , and this binding conferred high anti-28 S accessibility. These results indicated that the bindings of the P complex and L12 to their respective sites influenced the GTPase domain to increase the accessibility to anti-28 S. A possible RNA conformation adjusted by the protein bindings is discussed.Despite extensive evidence for functional importance of rRNA molecules within the ribosome, it has been difficult to demonstrate biological activities of protein-free rRNA under physiological salt condition. This difficulty appears to be due to involvement of ribosomal proteins that induce and stabilize the higher order structure of rRNA (reviewed by Noller, 1991). Detailed knowledge of rRNA-protein binding and its effect on the RNA conformation is, therefore, important to elucidate the molecular basis of rRNA function. The "GTPase domain" within domain II of Escherichia coli 23 S rRNA is one of the best characterized portions (reviewed by Cundliffe, 1986;Egebjerg et al., 1990;Ryan et al., 1991;Rosendahl and Douthwaite, 1993). This RNA region has been identified as a site of interaction with elongation factor G (Sköld, 1983;Moazed et al., 1988) and with the antibiotic thiostrepton, an inhibitor of elongation factor-dependent processes in protein synthesis (Thompson et al., 1982;Egebjerg et al., 1989). Ribosomal protein L11 and the acidic protein complex L10(L12) 4 cooperatively bind to this RNA domain (Dijk et al., 1979;Beauclerk et al., 1984) and construct a functional site of the 50 S subunit. L11 may participate in induction of a functionally important RNA conformation (Xing and Draper, 1995). On the other hand, the L10(L12) 4 complex appears to affect the RNA conformation through cooperative binding with L11 (Rosendahl and Douthwaite, 1993).The GTPase domain of eukaryotic 28 S rRNA has been also shown to interact with elongation factor 2 (Uchiumi and Kominami, 1994). However, its interaction w...

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Section: Conserved Protein Binding Features Of the Gtpase Domain-supporting

confidence: 59%

Binding of Mammalian Ribosomal Protein Complex P0·P1·P2 and Protein L12 to the GTPase-associated Domain of 28 S Ribosomal RNA and Effect on the Accessibility to Anti-28 S RNA Autoantibody

Uchiumi

Kominami

1997

Journal of Biological Chemistry

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“…Total ribosomal RNA from E. coli was prepared as described previously [2]. Ribosomal 23 S RNA was prepared by phenol extraction of 50s subunits as follows.…”

Section: Preparation Of Ribosomal R N Amentioning

confidence: 99%

“…Much of the information concerning this active site has been gathered by studying the interaction of the inhibitor thiostrepton with the larger (50 S) ribosomal subunit or with subparticles derived from it (for review, see [I]). Thus, the primary binding site for thiostrepton has been localized to 23 S rRNA within the region where protein L11 of the 50s ribosomal subunit also interacts [2]. Binding of the drug to 23s rRNA is relatively weak ( K d approximately 0.5 pM; M. Stark and E. Cundliffe, unpublished data) but is dramatically enhanced when protein L 11 is also complexed with the RNA.…”

mentioning

confidence: 99%

Studies of the GTPase domain of archaebacterial ribosomes

Beauclerk

Hummel²,

Holmes

et al. 1985

European Journal of Biochemistry

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Ribosomes from the methanogens Methanococcus vunnielii and Methanobacterium jormicicum catalyse uncoupled hydrolysis of GTP in the presence of factor EF-2 from rat liver (but not factor EF-G from Escherichiu coli). In this assay, and in poly(U)-dependent protein synthesis, they were sensitive to thiostrepton. In contrast, ribosomes from Sulfolohus solfataricus did not respond to factor EF-2 (or factor EF-G) but possessed endogenous GTPase activity, which was also sensitive to thiostrepton. Ribosomes from the methanogens did not support (p)ppGpp production, but did appear to possess the equivalent of protein L 11, which in E. coli is normally required for guanosine polyphosphate synthesis. Protein L 11 from E. coli bound well to 23 S rRNA from all three archaebacteria (as did thiostrepton) and oligonucleotides protected by the protein were sequenced and compared with rRNA sequences from other sources.Few functional domains of the eubacterial ribosome have been well characterized but one such is the GTPase centre whose activity is coupled to elongation factor G (EF-G). Much of the information concerning this active site has been gathered by studying the interaction of the inhibitor thiostrepton with the larger (50 S) ribosomal subunit or with subparticles derived from it (for review, see [I]). Thus, the primary binding site for thiostrepton has been localized to 23 S rRNA within the region where protein L11 of the 50s ribosomal subunit also interacts [2]. Binding of the drug to 23s rRNA is relatively weak ( K d approximately 0.5 pM; M. Stark and E. Cundliffe, unpublished data) but is dramatically enhanced when protein L 11 is also complexed with the RNA. As a result of such binding to native ribosomes, thiostrepton specifically inhibits factor-dependent GTP hydrolysis [3]. Further evidence that protein L l l participates in the ribosomal GTPase domain was forthcoming when this protein was labelled within the ribosome by a photoactivated derivative of GTP, in a reaction dependent upon the presence of factor EF-G [4]. Also, when factor EF-G was cross-linked to 70s ribosomes, protein L11 was again one of the targets [5], as was that specific portion of 2 3 s rRNA which is protected by protein L11 [ti].In addition to its involvement in GTPase activity, the domain of the 50s ribosomal subunit with which thiostrepton interacts also plays a role in the regulatory coupling of translation to a whole array of other cellular processes including the synthesis of rRNA, tRNA, ribosomal proteins and enzymes involved in amino acid biosynthesis (for review, see [7]). This occurs via the so-called 'stringent response' and involves the interaction of stringency factor with the aforementioned ribosomal domain. In response to the binding of uncharged tRNACorrespondence to E. Cundliffe, Department of Biochemistry, University of Leicester, Leicester, England LE 1 7 RH Abbreviations. Mc2S0, dimethylsulphoxide; EF-G, elongalion factor G ; EF-2, elongation factor 2; SDS, sodium dodecyl sulphate; ppGpp, guanosine 5'-diphosphate 3'-diphospha...

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“…Tight binding was expected from the fact that thiostrepton binds stoichiometrically to micromolar concentrations of ribosomes or L11-23 S rRNA complexes (45,46). The most likely origin of the cooperativity is direct interaction between the L11-NTD and thiostrepton, as suggested by a plausible structural model in which thiostrepton contacts both protein and RNA residues that are mutated in thiostrepton-resistant bacterial strains (19).…”

mentioning

confidence: 99%

Interactions of the N-terminal Domain of Ribosomal Protein L11 with Thiostrepton and rRNA

Bausch

Полякова

Draper

2005

Journal of Biological Chemistry

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Ribosomal protein L11 has two domains: the C-terminal domain (L11-C76) binds rRNA, whereas the N-terminal domain (L11-NTD) may variously interact with elongation factor G, the antibiotic thiostrepton, and rRNA. To begin to quantitate these interactions, L11 from Bacillus stearothermophilus has been overexpressed and its properties compared with those of L11-C76 alone in a fluorescence assay for protein-rRNA binding. The assay relies on 2-amino-butyryl-pyrene-uridine incorporated in a 58-nucleotide rRNA fragment, which gives ϳ15-fold enhancement when L11 or L11-C76 is bound.

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Binding of Thiostrepton to a Complex of 23‐S rRNA with Ribosomal Protein L11

Cited by 91 publications

References 10 publications

Binding of Mammalian Ribosomal Protein Complex P0·P1·P2 and Protein L12 to the GTPase-associated Domain of 28 S Ribosomal RNA and Effect on the Accessibility to Anti-28 S RNA Autoantibody

Binding of Mammalian Ribosomal Protein Complex P0·P1·P2 and Protein L12 to the GTPase-associated Domain of 28 S Ribosomal RNA and Effect on the Accessibility to Anti-28 S RNA Autoantibody

Studies of the GTPase domain of archaebacterial ribosomes

Interactions of the N-terminal Domain of Ribosomal Protein L11 with Thiostrepton and rRNA

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