Reconstitution of the 50 S ribosome subunit. Localization of activities related to the peptidyl transferase centre

Ballesta, Juan P. G.; Montejo, Vicente; Vázquez, David

doi:10.1016/0014-5793(71)80609-9

Cited by 17 publications

(6 citation statements)

References 15 publications

(13 reference statements)

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“…Discussion Acknowledgements The G-dependent GTPase activity of the ribosomes requires the proteins involved in the /3-to y-core step. A similar requirement has been shown for a number of activities of the peptidyl transferase centre [6]. However, the GTPase centre of the 50 S subunit is unique among those so far studied [4-61, requiring at least partially the proteins released in the conversion of 50 S subunits to cy-cores for its normal function.…”

Section: Tim Of Incubationmentioning

confidence: 65%

“…Ribosomes and ribosome subunits were prepared from E. coli D-10 as previously described [6] . a-, p-and y-cores and two types of split proteins SP,,_p and SPe_, (released in the conversion of 50 S to p-cores and /3-to y-cores respectively) were obtained by isopycnic centrifugation in CsCl gradients [3].…”

Section: Ribosomes Ribosome Subunits and Derived Particlesmentioning

confidence: 99%

“…1 which presents the kinetics of the reaction carried out by the various particles. Whereas in studies on the peptidyl transferase activity, o-cores show a higher activity than the 50 S subunits from which they were derived [4,6], this is not the case when the G-dependent GTPase is studied. y-cores, upon prolonged incubation, display a low but reproducible GTPase activity.…”

Section: G-dependent Gtpase Activity Of 50 S Derived Particlesmentioning

confidence: 99%

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Reconstitution of the 50 S ribosome subunit. Localization of G‐dependent GTPase activity

Ballesta¹,

Montejo²,

Vázquez³

1971

FEBS Letters

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Section: Tim Of Incubationmentioning

confidence: 65%

Section: Ribosomes Ribosome Subunits and Derived Particlesmentioning

confidence: 99%

Section: G-dependent Gtpase Activity Of 50 S Derived Particlesmentioning

confidence: 99%

See 1 more Smart Citation

Reconstitution of the 50 S ribosome subunit. Localization of G‐dependent GTPase activity

Ballesta¹,

Montejo²,

Vázquez³

1971

FEBS Letters

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“…Experiments on the resistance of nascent polypeptide chains to proteolysis in eukaryotes (16,17) and in Bacillus subtilis (18) have demonstrated that the carboxyl terminus ofthe chain is protected from degradation. This region of the chain contains the most recently synthesized 30-40 residues (30)(31)(32)(33)(34)(35)39, and 30 residues in refs. 16,17, and 18, respectively).…”

Section: Discussionmentioning

confidence: 99%

Nascent polypeptide chains emerge from the exit domain of the large ribosomal subunit: immune mapping of the nascent chain.

Bernabéu¹,

Lake²

1982

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

128

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The site of the nascent polypeptide chain as it leaves the ribosome has been localized on the "exit domain" of the Escherichia coli ribosome by using IgG antibodies directed against the enzyme 13-galactosidase (EC 3.2.1.23). Thus, a functional site has been mapped on intact 70S ribosomes. The exit site is on the large subunit, approximately 70 A from the interface between subunits and nearly 150 A from the central protuberance, the likely site of peptide transfer. It is adjacent to the region corresponding to the rough endoplasmic membrane binding region ofthe eukaryotic ribosome but distant from ribosomal components participating in mRNA recognition and polypeptide elongation (ie., distant from the "translational domain"). These results, together with the protease protection experiments of others, provide evidence that the nascent protein chain probably passes through the ribosome in an unfolded, fully extended conformation.The ribosome, as the organelle that translates genetic messages into proteins, has roles both in the translation of the message and in the manufacture and secretion ofthe protein. Currently, a good deal is known about the biochemistry of ribosomal components and factors, but this knowledge is only now being integrated with that of ribosomal three-dimensional structure. Although the locations of many of the ribosomal components involved in the translation ofthe code are known, little is known about the location and path of the protein chain as it passes through the ribosome. Those sites functioning in translation are clustered into part of the ribosome, composing approximately two-thirds of its volume, that we have named the "translational domain. " Functional sites contained in the translational domain include: the initiation factor binding sites (1-4) located in the cleft of the small subunit, the messenger binding sites located on the platform of the small subunit (3,(5)(6)(7), the peptidyltransferase and the 5S RNA located on the central protuberance of the large subunit (8)(9)(10)(11), and proteins mediating the GTP-dependent steps of translation that are found on the L7/L12 stalk of the large subunit (12). Together, these sites define the translational domain.In this paper, we have investigated the location ofthe nascent chain as it emerges from the ribosome, a function associated with protein secretion rather than the translation steps just described. Using antibodies directed against the enzyme ,B-galactosidase (EC 3.2.1.23) to map the exit site ofthe nascent protein chain, we find that it exits from the ribosome at a single region, located on the large subunit. This region is distant from the translational domain and is approximately 150 A from the presumed site of the peptidyltransferase (8,9). No functional site has been mapped directly on 70S ribosomes previously, and in addition the site is found on a region of the 50S subunit where no proteins or functions have been previously found. The corresponding region of the eukaryotic ribosome (13, 14), however, contains a ribos...

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“…Polyphenylalanine synthesis, EF G-dependent hydrolysis of GTP and peptide bond formation (peptidyl transferase activity) in the fragment reaction assay were carried out as previously described10 '14* Unless otherwise stated in the text, buffer and ion concentrations in the reaction mixtures were 80 mM NH4Cl, 10 mM Mg acetate, 20 mM Tris-HC1 (pH 7.8) and 1 mM dithiothreitol (DTT). Treatment of the ribosomes (1 mg/ml) with ethidium bromide was carried out in 20 mM Tris-HCl (pH 7.8), containing 100 mM NH4Cl and 1 mM or 10 mM MgCl2 (buffer A).…”

Section: Methodsmentioning

confidence: 99%

Specific release of ribosomal proteins by nucleic acid-intercalating agents.

Ballesta¹,

Waring²,

Vázquez³

1976

Nucleic Acids Research

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Increasing concentrations of ethidium bromide cause progressive inactivation of ribosomes, apparently by binding to double-stranded regions of the rRNA. At low drug concentrations (10(-4)M) the partial inhibition detected is due to specific release of proteins L7 and L12; activity can be restored by addition of an excess of these two proteins. At higher concentrations the inactivation is not reversed by supplementation with released proteins. The presence of ethanol affects the extent of ethidium binding and also the release of ribosomal proteins. In all tests the proteins most sensitive to the presence of the drug are L7 and L12, followed by L8/9, L11, L27, L28, L29 and L30. Despite the fact that L7 and L12 are the first two proteins released by ethidium they are never totally missing from drug-treated ribosomes, though the other proteins can be displaced completely. About 50% of proteins L7 and L12 remain on the ribosomes at the highest drug concentrations tested, possibly indicating heterogeneity in the binding sites for the several copies present in the ribosome.

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Reconstitution of the 50 S ribosome subunit. Localization of activities related to the peptidyl transferase centre

Cited by 17 publications

References 15 publications

Reconstitution of the 50 S ribosome subunit. Localization of G‐dependent GTPase activity

Reconstitution of the 50 S ribosome subunit. Localization of G‐dependent GTPase activity

Nascent polypeptide chains emerge from the exit domain of the large ribosomal subunit: immune mapping of the nascent chain.

Specific release of ribosomal proteins by nucleic acid-intercalating agents.

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