Polypeptide synthesis with ribonuclease-digested ribosomes

Cahn, Frederick; Schachter, Eugene M.; Rich, Alexander

doi:10.1016/0005-2787(70)90748-3

Cited by 53 publications

(15 citation statements)

References 13 publications

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“…That the ribosome is a compact essentially impenetrable particle is shown by the inaccessibility of about half of its constituents to water, as evidenced by the hydrogen exchange studies of Page et al [24]. It is very unlikely that a large proportion of the protein is displaced by the nuclease to expose RNA because after digestion to the extent observed in our experiments, no proteins are found in the supernatant (as judged by the Folin-Ciocalteu reaction), and all interaction sites are evidently intact, for the particles sediment as intact ribosomes, and are known [7] to be biologically functional.…”

Section: Discussionsupporting

confidence: 54%

“…There have subsequently been many studies on the digestion of ribosomes with nucleases [2-61, and in one of the most recent [7], it has been reported that a considerable part of the RNA can be detached without elimination of biological activity. It has been found in this laboratory and elsewhere [8-121 that the controlled attack of nucleases on ribosomal RNA produces a series of sharply defined fragments which can readily be separated by polyacrylamide gel electrophoresis and can serve as a kind of fingerprint of an RNA species [12].…”

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confidence: 99%

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Nuclease Degradation and the Structure of Ribosomes

Pinder¹,

Gratzer²

1972

European Journal of Biochemistry

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Well-defined patterns of fragments are produced when Escherichia coli ribosomes, both native, dissociated, unfolded, and partially denuded of proteins by treatment with caesium chloride, are digested with pancreatic ribonuclease, and the extracted RNA is fractionated by polyacrylamide gel electrophoresis. Procedures are given for determining the degree of congruence of a pair of digestion patterns, and analysing the results in terms of probability of matching. By this means it has been established that all the digestion patterns which have been obtained are closely related. The similarity between native 70-8 ribosomes and completely unfolded nucleoprotein particles in EDTA indicates that the greater part of the RNA is accessible on the surface of the 70-S ribosome. It has not been possible to show a close relationship between the patterns generated by the nucleoprotein particles in various states and these from free ribosomal RNA. We infer that a number of the labile points in the RNA are protected by bound proteins. The digestion proceeds much more slowly with increasing stabilisation of structure by magnesium ions, both in free RNA and ribonucleoprotein. It is shown that the labile points of the chain are segments of some length, and not isolated phosphodiester links.The availability of RNA in the 70-S ribosome of Escherichia coli to pancreatic ribonuclease was first noted by Xanter [I], who deduced that the RNA is on the surface of the particle, but is in a more resistant state than free, extracted RNA. There have subsequently been many studies on the digestion of ribosomes with nucleases [2-61, and in one of the most recent [7], it has been reported that a considerable part of the RNA can be detached without elimination of biological activity. It has been found in this laboratory and elsewhere [8-121 that the controlled attack of nucleases on ribosomal RNA produces a series of sharply defined fragments which can readily be separated by polyacrylamide gel electrophoresis and can serve as a kind of fingerprint of an RNA species [12]. Although the lability of the RNA to nuclease is undoubteldy affected by basepairing, our evidence [Ill on digestion with T I ribonuclease points to a specificity of cleavage determined by primary structure, for essentially the same pattern of zones can be obtained from an RNA from which base-pairing has been eliminated by treatment with formaldehyde. The digestion pattern is thus a characteristic of the RNA chain, and one might hope to be able to use it in such a structure as the ribosome to establish to what extent the limited number of primary labile regions of the RNA are available to the enzyme in various conformational states, The results ofsuch a study are presented here. MATERIALS AND METHODS Preparation of RibosomesRibosomes from nuclease-deficient strain MRE 600 of E . coli were prepared by standard procedures [13], and RNA in general by extraction with phenol and detergent [14]. Run-off and "stuck" ribosomes, which differ in that the former contain no messenger, and are ...

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Section: Discussionsupporting

confidence: 54%

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confidence: 99%

Nuclease Degradation and the Structure of Ribosomes

Pinder¹,

Gratzer²

1972

European Journal of Biochemistry

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“…As known, 23 S RNA within the 50 S subparticle of E. coli ribosomes apparently has 3 exposed sites which are most sensitiye to the action of different ribonucleases specific to single-stranded regions of the polynucleotide chain (pancreatic RNase [21][22][23][24][25][26][27][28][29], TI-RNase [30] and endogenous ribosomal RNase from E. co& [26]). One of these sites (site 1, see scheme in fig.1) is positioned at a distance of about 1171-l 178 nucleotides from the 5 '-end of 23 S RNA [31,32] and its hydrolysis gives two fragments detected by SDS-PAGE without denaturation of the RNA secondary structure: the 3 '-terminal 18 S and 5 '-terminal 13 S fragments .…”

Section: Hy~ro~ysjs Of the 50 S S~~p~r~~c~e With ~Nusementioning

confidence: 99%

Elongation factor G protects a nuclease‐sensitive site of 23 S RNA within the ribosome

Bochkareva

Girshovich

1984

FEBS Letters

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“…Transition elements have striking effects on the tertiary structure of RNA molecules (4,12) which are over and above the effects of hydrogen bonding (9). Hydration (29), unfolding (35,36,37), and swelling (38) (3,11,31,40), and the fragmented RNA was not released from the ribosomes (3,31 …”

Section: Discussionmentioning

confidence: 99%

A Role for Zinc in the Structural Integrity of the Cytoplasmic Ribosomes of Euglena gacilis

Prask

Plocke

1971

Plant Physiol.

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Zinc deficiency in dark-grown Euglena gracilis Klebs, Z strain Pringshein, results in the disappearance of cytoplasmic ribosomes. In contrast, ribosomes in zinc-sufficient Euglena are conserved, do not undergo turnover, and can be demonstrated at any stage of growth. The zinc content of ribosomes from zincdeficient Euglena just prior to ribosomal disappearance is 300 to 380 micrograms of zinc per gram rRNA as compared to 650 to 1280 micrograms of zinc per gram rRNA in ribosomes from zinc-sufficient cells. Ribosomal disappearance is believed to involve a generalized disintegration process related to the lower content of zinc in the ribosomes. Reappearance of ribosomes requires the addition of zinc. It is proposed that adequate zinc may be essential for nornal tertiary and quaternary structure of the cytoplasmic ribosomes of Euglena.

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Polypeptide synthesis with ribonuclease-digested ribosomes

Cited by 53 publications

References 13 publications

Nuclease Degradation and the Structure of Ribosomes

Nuclease Degradation and the Structure of Ribosomes

Elongation factor G protects a nuclease‐sensitive site of 23 S RNA within the ribosome

A Role for Zinc in the Structural Integrity of the Cytoplasmic Ribosomes of Euglena gacilis

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