Structural transformations of ribosomes (dissociation, unfolding and disassembly)

Spirin, Alexander S.

doi:10.1016/0014-5793(74)80687-3

Cited by 39 publications

(14 citation statements)

References 166 publications

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“…The COOHterminal domain of ribosomal protein L11 excludes 1700 to 1900 A2 from solvent when it binds to 23s rRNA (11,12,57). Considering the size of the ribosome, the relatively small surface area involved in intersubunit contacts is consistent with the delicate balance between subunit association and dissociation observed in vitro (58).…”

Section: Because the Asl (Gray) Is Common T O Both Ribosome Complexessupporting

confidence: 60%

X-ray Crystal Structures of 70 S Ribosome Functional Complexes

Cate

Yusupov

Yusupova

et al. 1999

Science

569

409

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Structures of 70S ribosome complexes containing messenger RNA and transfer RNA (tRNA), or tRNA analogs, have been solved by x-ray crystallography at up to 7.8 angstrom resolution. Many details of the interactions between tRNA and the ribosome, and of the packing arrangements of ribosomal RNA (rRNA) helices in and between the ribosomal subunits, can be seen. Numerous contacts are made between the 30S subunit and the P-tRNA anticodon stem-loop; in contrast, the anticodon region of A-tRNA is much more exposed. A complex network of molecular interactions suggestive of a functional relay is centered around the long penultimate stem of 16S rRNA at the subunit interface, including interactions involving the "switch" helix and decoding site of 16S rRNA, and RNA bridges from the 50S subunit.

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Section: Because the Asl (Gray) Is Common T O Both Ribosome Complexessupporting

confidence: 60%

X-ray Crystal Structures of 70 S Ribosome Functional Complexes

Cate

Yusupov

Yusupova

et al. 1999

Science

569

409

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“…The effect of magnesium on the sedimentation pattern of the SucNBr-stimulated preparation might be explained by an increase in affinity between the modified subunits promoted by magnesium. This effect is similar to that on the sedimentation pattern of untreated ribosomes [20], the difference being the lower affinity between the modified subunits as compared with that between those untreated.…”

Section: Discussionsupporting

confidence: 77%

Stimulation of Polypeptide Polymerization in Escherichia coli Ribosomes by Modification of Ribosomal Sulfhydryl Groups with N‐Bromosuccinimide

Lópbz‐Rivas

Vázquez

Palacián

1978

European Journal of Biochemistry

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Modification of Escherichiu coli ribosomes with N-bromosuccinimide (SucNBr) (molar ratio of reagent to ribosome equal to 10) is accompanied by 100 -200 %increase in poly(U)-directed polyphenylalanine synthesis, while no change in peptidyl transferase activity ('fragment reaction') was observed. The magnesium dependence of the modified ribosomes in polyphenylalanine synthesis (optimum at 11 mM) is different from that of untreated ones (optimum at 9 mM). Hybridization of SucNBr-treated and untreated subunits showed that stimulation is produced by modification of the 50-S subunit.Stimulation is the result of an increase in the rate of polyphenylalanine synthesis without activation of inactive ribosomes present in the preparation. Treatment with SucNBr is also accompanied by the appearance of a peak intermediate between 70-S ribosomes and 5 0 3 subunits in sucrose gradient centrifugation. At 7 mM magnesium the intermediate peak appears closer to 50-S subunits than at 10 mM, disappearing at 20 mM magnesium or after treatment with glutaraldehyde with a concomitant increase in 70-S ribosomes. These results suggest that the observed intermediate peak is produced by a rapid association/dissociation equilibrium between modified 50-S and 30-S subunits with decreased affinity for each other. Modification with 5,5'-dithio-bis(2-nitrobenzoate) (Nbsz) prior to the SucNBr treatment prevents the stimulation of polyphenylalanine synthesis. When the SucNBr-stimulated ribosomes are incubated with 2-mercaptoethanol or dithiothreitol the observed stimulation disappears. These results indicate that stimulation is produced by modification of ribosomal sulfhydryl groups by SucNBr. The stimulatory modification with SucNBr decreases by 5 -6 the number of -SH groups per ribosome reactive with Nbsz while no significant change in the total number of tryptophan residues was observed (tryptophan residues are the next most likely reactive groups in the ribosome).Much work has been done on the chemical modification of ribosomal sulfhydryl groups and its effects on the functional and structural properties of the ribosome. However, the results obtained are sometimes confusing and even contradictory. Treatment of Escherichia coli ribosomes with sulfhydryl-group reagents is usually accompanied by inactivation of polypeptide polymerization. In some cases inactivation reaches 30-70% and does not increase any more with further increases in reagent concentration or incubation time [1,2]. According to Moore [2], a fraction of ribosomes is susceptible to inactivation while the rest are resistant. The inactivation is located at the 30-S subunit [l]. Modification by most sulfhydrylAhhreviutions. HOHgBzOH, p-hydroxymercuribenzoate ; Nbsz, Enzyme. Pyruvate kindse (EC 2.7.1.40). (2-nitrobenzoate); SucNBr, N-bromosuccinimide. group reagents is followed by dissociation of ribosomes into 50-S and 30-S subunits, and sometimes by the appearance of a component with a sedimentation rate intermediate between 70 S and 50 S [1,3,4]. Dissociation is not observed at high ...

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“…Chelating agents such as EDTA have been successfully employed to partially unfold a number of RNPs including ribosomal subunits, RNase P, small cytoplasmic RNPs and SRP, which were subsequently disassembled into protein and RNA components by column chromatography (Blobel 1971;Spirin 1974;Newton et al 1975;Guthrie and Atchison 1980;Mukherjee and Sarkar 1981;Walter and Blobel 1983). To address the effect of EDTA on reconstitution of telomerase activity, we first treated partially purified active telomerase fractions with Ca^"^ and MNase to remove endogenous telomerase RNA.…”

Section: Reconstitution Of Telomerase Activity After Mnase Digestionmentioning

confidence: 99%

Functional reconstitution of wild-type and mutant Tetrahymena telomerase.

Autexier¹,

Greider²

1994

Genes Dev.

100

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Telomerase is a ribonucleoprotein that catalyzes telomere elongation in vitro and in vivo. The 159-nucleotide RNA component of Tetrahymena telomerase contains the sequence 5'-CAACCCCAA-3' ("template region"), which serves as a template for the addition of the sequence d(TTGGGG)" to Tetrahymena telomeres. To dissect the Tetrahymena telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential telomerase RNA by micrococcal nuclease digestion of partially purified telomerase, the addition of in vitro-transcribed telomerase RNA reconstituted telomerase activity. The reconstituted activity was processive and showed the same primer specificities as native telomerase. Mutants in the RNA template region were tested in reconstitution assays to determine the role of the residues in this region in primer recognition and elongation. Two template mutants, encoding the sequences 5'-UAACCCCAA-3' and 5'-UAACCCUAA-3', specified the incorporation of dATP into the sequence d(TTAGGG). Telomerase reconstituted with a template mutant encoding the sequence 5'-CAACCCUAA-3' did not specify dATP incorporation and elongation by this mutant was not terminated by the addition of ddATP. In addition, a template mutant encoding the sequence 5'-CGGCCCCAA-3' specified the incorporation of ddCTP but not ddTTP while a mutant encoding the sequence 5'-CAACCCCGG-3' specified the incorporation of ddTTP but not ddCTP. These data suggest that only the most 5' six residues of the template region dictate the addition of telomeric repeats.

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Structural transformations of ribosomes (dissociation, unfolding and disassembly)

Cited by 39 publications

References 166 publications

X-ray Crystal Structures of 70 S Ribosome Functional Complexes

X-ray Crystal Structures of 70 S Ribosome Functional Complexes

Stimulation of Polypeptide Polymerization in Escherichia coli Ribosomes by Modification of Ribosomal Sulfhydryl Groups with N‐Bromosuccinimide

Functional reconstitution of wild-type and mutant Tetrahymena telomerase.

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