Structural studies on Escherichia coli ribosomes: III. Denaturation and sedimentation of ribosomal subunits unfolded in urea

doi:10.1016/0005-2787(70)90707-0

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis

Volume 199, Issue 1, 21 January 1970, Pages 184-193

https://doi.org/10.1016/0005-2787(70)90707-0 Get rights and content

Abstract

The addition of urea to 50-S ribosomal subunits caused a lowering of the sedimentation coefficient to 32–38 S by a one-step co-operative process. This was interpreted as an unfolding of the native ribosome caused by the disruption of hydrophobic bonds between ribosomal proteins. In many cases slow-moving components were evident in the ultracentrifuge which were probably dissociated protein.

Treatment of 50-S ribosomes with formaldehyde prevented the dissociation of the protein from RNA on subsequent reaction with urea. The ribosomes appeared to unfold to give slower-sedimenting species but the nature of the unfolding was different from that of untreated ribosomes. Thus the ribosomes were more sensitive to reaction with urea and the unfolding was no longer highly co-operative.

The addition of varying amounts of EDTA to formaldehyde-treated ribosomes produced a single unfolded component with a sedimentation coefficient of 34–38 S. Unlike untreated ribosomes no further unfolding occurred, either with time or with a large excess of EDTA.

No loss of secondary structure in the RNA occurred when native or formaldehyde-treated ribosomes were reacted with urea or EDTA at room temperature. The melting temperatures decreased in urea and EDTA and the melting curves became less co-operative. The melting profiles of unfolded ribosomes in urea and EDTA were similar to RNA in the same solvent and showed that in the unfolded form the protein had little effect on the thermal denaturation properties of the RNA. It is concluded that in the unfolded ribosomes, characterised by a sedimentation coefficient of 30–35 S, the major bonds involved in the maintenance of ribosomal tertiary structure have been broken. These bonds involve interactions between the proteins of the ribosome.

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Cited by (13)

Characterization of the particles produced by exposure of ribosomal subunits to urea
1975, BBA Section Nucleic Acids And Protein Synthesis
When Escherichia coli 50-S ribosomal subunits are treated with increasing concentrations of urea partial deproteination occurs. Furthermore, we observed that the number of sulfhydryl groups which react with Ellman's reagent is a sigmoidal function of the urea concentration. These results are similar to those previously reported for the 30-S subunit (Acharya, A.S. and Moore, P.B. (1973) J. Mol. Biol. 76, 207–221). For both subunits we identify the proteins which dissociate (split proteins) or are recoverable in a ribonucleoprotein particle (core proteins) under the action of 6 M urea in a buffer of moderate ionic strength.
Structural transformations of ribosomes (dissociation, unfolding and disassembly)
1974, FEBS Letters
Degradation of ribonucleic acid in rat liver ribosomes
1973, Journal of Molecular Biology
The enzymatic degradation of RNA in rat liver ribosomes has been investigated using pancreatic, T₁ and rat liver ribonucleases. The following results have been obtained.
- 1.
  (1) Ribosomes, prepared with Triton X100, precipitated and washed with 0.1 m-magnesium ions, do not exhibit detectable ribonuclease activity over long incubation periods. This made it possible to study the action of known amounts of different ribonucleases.
- 2.
  (2) During ribonuclease digestion, the rate of formation of acid-soluble products falls exponentially, so that degradation almost reaches a saturation state characteristic for the enzyme concentration.
- 3.
  (3) The sensitivity of RNA in situ to pancreatic ribonuclease is increased by EDTA, even in particles fixed with formaldehyde, where dissociation into sub-particles and unfolding are prevented. Heating the ribosomes with formaldehyde also increases the sensitivity of RNA, probably by converting it into a single-stranded form. In such a state, RNA seems to protect the ribosomal proteins against pronase.
- 4.
  (4) Conditions have been defined for obtaining highly reproducible digestion patterns of RNA fragments.
- 5.
  (5) It has been found that the stability of the phosphodiester bonds in RNA in situ varies over a rather wide range (3 to 4 orders of magnitude). The distribution of labile sites along RNA is highly irregular: while 20% of the RNA has been converted into acid-soluble products, fragments about 500 nucleotides long remain completely intact.
- 6.
  (6) It has been shown that the pancreatic, t₁ and rat liver ribonucleases attack RNA in situ at a similar set of sites. This suggests that the specificity of the breakdown is determined mainly by the quaternary structure of the ribosomal particles.
Reactions of Nucleic Acids and NucleoDroteins with Formaldehyde
1973, Progress in Nucleic Acid Research and Molecular Biology
This chapter discusses the three main reasons for the chemical modification of nucleic acids that are available on modifying agents. These agents are used for inactivations of various kinds, for directed functional changes of nucleic acids in vivo and for the elucidation, through resulting modification, of structural and functional characteristics peculiar to synthetic or native polynucleotides. Some of these agents are useful for one, two, or all three of these purposes. One of the few agents that serve all three of them is formaldehyde. It is widely used as an inactivator of viruses to obtain vaccines and is reported to exert a cytostatic (carcinostatic) effect. It is also one of the most promising mutagenic agents affecting multicellular organisms. Formaldehyde is used extensively in structural and functional studies of nucleic acids as an agent not so much for causing denaturation as for preventing renaturation, and as a fixator of nucleic acids and nucleoproteins in electron microscope and sedimentation investigations. Recently, some progress in the structural study of formaldehyde interaction products with nucleotides and nucleic acids has been made. The chapter emphasizes on the evidence for the formation of various structures and the molecular mechanisms of biological and other effects of formaldehyde.
On the mode of reaction of formaldehyde with ribosomes
1972, BBA Section Nucleic Acids And Protein Synthesis
Formaldehyde treatment of compact and unfolded ribosomes prevents dissociation into protein and rRNA by a variety of agents known to induce dissociation of untreated particles. This stabilization derives from inter- or intramolecular protein-protein crosslinks rather than protein-rRNA crosslinks. Intermolecular protein-protein crosslinking is enhanced in the native, compact state as compared to the denatured, unfolded state.
rRNA regions resistant to nuclease digestion of unfolded ribosomes are also protected in situ against chemical modification by formaldehyde. On the other hand RNA fragments obtained through nuclease digestion of isolated rRNA derive from RNA regions freely accessible to formaldehyde. The results support that digestion of ribosomes yields RNA fragments which are representative for the protein binding sites in situ.
Nascent Chains Derived from a Foldable Protein Sequence Interact with Specific Ribosomal Surface Sites near the Exit Tunnel
2023, Research Square

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Structural studies on Escherichia coli ribosomes: III. Denaturation and sedimentation of ribosomal subunits unfolded in urea

Abstract

Biochim. Biophys. Acta

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Biochem. Biophys. Res. Commun.