Thermodynamics of RNA unfolding: Stabilization of a ribosomal RNA tertiary structure by thiostrepton and ammonium ion

Draper, David E.; Xing, Yanyan; Laing, Lance G.

doi:10.1006/jmbi.1995.0291

Cited by 39 publications

(34 citation statements)

References 23 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-mentioning

confidence: 55%

“…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).…”

mentioning

confidence: 99%

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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-mentioning

confidence: 55%

mentioning

confidence: 99%

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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“…Nuclease protection and fragment binding studies have defined the sequence 1051-1108 as the principal protein recognition site (6,12,17,18) (11,13,19 …”

Section: Resultsmentioning

confidence: 99%

“…RNA fragment alone binds thiostrepton normally (11,19). The remaining 17 hits are concentrated in a few selected regions ofthe molecule and all of these can be rationalized as mutations that either stabilize or do not affect the RNA structure.…”

Section: Discussionmentioning

confidence: 99%

“…Ligand binding and RNA melting experiments also show that LII and thiostrepton specifically stabilize the NH4+-dependent tertiary structure (10)(11)(12). Thus LII and thiostrepton must both recognize the RNA tertiary structure at some level, though mutations disrupting the tertiary structure have a much larger effect on thiostrepton binding affinity than on LII affinity (13).…”

Section: Introductionmentioning

confidence: 96%

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On the role of rRNA tertiary structure in recognition of ribosomal protein L11 and thiostrepton

Draper

1995

Nucl Acids Res

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Rapid Magnesium Chelation as a Method to Study Real‐Time Tertiary Unfolding of RNA

Maglott

Glick

2001

CP Nucleic Acid Chemistry

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This unit describes a method to measure the unfolding of RNA tertiary structure on a millisecond time scale. A stopped-flow spectrophotometer is used to measure the rate of unfolding induced by the addition of EDTA to an RNA whose tertiary structure has been stabilized in the presence of magnesium ions. Using this methodology, rate constants for unfolding of tertiary or secondary structure can be obtained over a range of temperatures, and these values can be used to construct Arrhenius and Eyring plots, from which activation energy, Arrhenius pre-exponential factor, and enthalpy and entropy of activation can be obtained. These data provide information about the energy of the transition state and the energy barriers between secondary and tertiary structure, which is necessary for predicting RNA tertiary structure from secondary structure.

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Thermodynamics of RNA unfolding: Stabilization of a ribosomal RNA tertiary structure by thiostrepton and ammonium ion

Cited by 39 publications

References 23 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

On the role of rRNA tertiary structure in recognition of ribosomal protein L11 and thiostrepton

Rapid Magnesium Chelation as a Method to Study Real‐Time Tertiary Unfolding of RNA

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