The Influence of 6‐M Urea on 30‐S Ribosomes of Escherichia coli

Reductive methylation of rat liver ribosomal subunits was carried out at different temperatures and the reaction was followed as a function of time. The percentage of protein lysine residues which were methylated reached a definite plateau for each temperature, and was considerably increased by heating (four times at 40 "C). This increase was not observed when free ribosomal proteins were heated under the same conditions. Ribosomal subunits kept their biological activity (elongation steps of protein synthesis) even when methylated to a high extent. Half of the subunit activity was still found when 67 % of the lysine residues were methylated.Proteins were divided into different classes on the basis of their alkylation in response to temperature. Those which reacted poorly within subunits even at high temperature should interact directly with rRNA. The results suggest that the protein reactivity modifications induced by heating (up to 37 "C), which were often totally or partially reversible, reflect a conformational change of the proteins themselves within the subunits. Besides protein unfolding, higher temperatures produced structural modification and inactivation of the subunits, which were restrained by the presence of aminoacyltRNA.Chemical modifications of ribosomes with protein reagents, which have often been used to study the accessibility of ribosomal proteins on the organelle, are also a very useful tool for studying conformational changes of ribosomal subunits. Among the numerous chemical reactions already carried out on bacterial ribosomes, reductive alkylation is of particular interest, because it is a very mild method ; in particular it makes possible the preparation of labelled ribosomes that remain functionally active [l]. In a previous study [2], we analysed the properties of eucaryotic ribosomes labelled by this method. It was observed that ribosomes kept all their activity in polyphenylalanine synthesis after this treatment, and that all the ribosomal proteins were alkylated to a varying but very low extent.In this communication, it is shown that the number of reactive lysine residues considerably increases if ribosomal subunits are heated during methylation (instead of being treated by formaldehyde and borohydride at 4 "C as in all the experiments mentioned above). This temperature effect was not observed when free proteins were heated. Therefore the method was used to study the conformational change of subunits induced by heating, the reversibility of this change and the protective effect of tRNA binding. MATERIALS AND METHODS Ribosomes und SubunitsPreparation of ribosomes and separation of active 40-S and 60-S subunits was performed as previously described [3]. The subunits were stored in buffer A (1 mM potassium phosphate pH 7.3, 30 mM KCl, 1.5 mM MgC12, 20 mM 2-mercaptoethanol). Reductive MethylationThe reaction was carried out in a fume cupboard as follows. Ribosomes or ribosomal subunits (1 mg of protein/ml) were suspended in buffer A containing 20 mM sodium borate, the pH of this buffer b...

Section: Discussionmentioning

confidence: 99%

Change of Protein Reactivity in Mammalian Ribosomal Subunits as a Function of Temperature

Reboud

Buisson

Marion

et al. 1977

“…After ultraviolet irradiation and ethanol precipitation, the 30-S subunits were treated with 6 M urea in a 10 mM Tris-HC1 pH 7.5, 330 mM magnesium acetate buffer, in order to release the non-crosslinked proteins from the 16-S RNA, according to SpitnikElson et al [30]. After 6 h treatment in the ice, the subunits were centrifuged at 0.2 mg/ml in the above buffer in a Spinco SW50 rotor at 45000 rev./min for 20 h.…”

Section: Release Of the Non-crosslinked Proteins From The Irradiated mentioning

confidence: 99%

Effect of Ultraviolet Irradiation on 30‐S Ribosomal Subunits. Identification of the RNA Region Crosslinked to Protein S7

Ehresmann

Backendorf

Ehresmann

et al. 1980

The effects of ultraviolet irradiation on Escherichia coli 30-S ribosomal subunits were studied. At the doses of radiation used in this work (0-4.5 x lo5 quanta/30-S subunit), only protein S7 was found to be significantly crosslinked to the 16-S RNA. In conditions where 25 % of the protein was covalently crosslinked, the ability of the irradiated 30-S subunits to reassociate with 50-S subunits and their activity in polyphenylalanine synthesis decreased strongly. Similar results were obtained by irrradiation with a germicide lamp (254 nm) or with a monochromatic ultraviolet light at 248 nm. No additional proteins were crosslinked to the 16-S RNA by irradiating 30-S subunits depleted in protein S1 or 70-S ribosomes.The covalent complex of 16-S RNA and protein S7 was isolated and digested by TI ribonuclease.

“…Whereas essentially no loss of secondary structure in the ribosomal RNA occurred upon exposure to urea [36,37] a remarkable change was observed for the particle RNA (Fig. 4).…”

Section: Sodium C L O T / C~c ; I~l S U L F E I~e ~~O L~~c I T~imentioning

confidence: 99%

“…As in the case of ribonucleoprotein particles, where urea treatment released a specific set or proteins (Fig. 2), several distinct categories of proteins are dissociated from the ribosomes [37,38]. Although the pattern of proteins released from ribosomes by exposure to urea differed from that obtained after high salt, a considerable overlap in protein composition between salt and urea-treated particles has been observed [38].…”

Section: Sodium C L O T / C~c ; I~l S U L F E I~e ~~O L~~c I T~imentioning

confidence: 99%

The Effect of Urea on the Structure of Nuclear Ribonucleoprotein Particles from Rat Liver

Northemann

Klump

Heinrich

1979

Ribonucleoprotein particles were isolated in the presence of cytosolic RNase inhibitor from rat liver nuclei and exposed to increasing concentrations of urea. The average sedimentation coefficient of 90 S in a sucrose gradient decreased slightly with urea concentrations of up to 2 M. At 5 M urea the particles sedimented at about 20 S. The protein-to-RNA ratio decreased from 4 to 1.4 after treatment with 5 M urea. The proteins which are preferentially detached from the ribonucleoprotein complex are in the molecular weight range of 30000-45000. In addition, small nuclear RNAs (snRNA) were dissociated from the ribonucleoprotein particles by high urea concentrations (> 2 M). From the fact that snRNAs are dissociated from the ribonucleoprotein complex by high urea concentrations and not dissociated by high NaCl concentrations even after extensive proteolytic digestion, it is concluded that hydrogen bonds are involved in their binding to heterogeneous nuclear RNA (hnRNA).The change in the RNA conformation after addition of increasing concentrations of urea was studied by circular dichroism measurements of ribonucleoprotein particles and protein-free particle RNA. In the presence of 5 M urea the molar ellipticities at 264 nm decreased from 15