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

Abstract

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...

Characterization and properties of a 5S‐RNA‐protein complex released from heated 60S ribosomal subunits

“…The sharp peaks at the left of the tracings (a) and (b) represent ribosomal subunits that could not penetrate into the gels. Arrows indicate the place of pure RNAs used markers alanine/puromycin reaction [21,22]. This conversion is concomitant with a disruption of hydrogen bonds within the subunits, a fact deduced from the melting curve, and with the release of 5s RNA 1221.…”

Photo‐Induced Protein‐RNA Cross‐Linking in Mammalian 60‐S Ribosomal Subunits

“…Under these preincubation conditions, only reversible unfolding of proteins took place [19]. Furthermore heating at 35 "C broke more heat-labile protein-RNA links than at 25 "C and, from the above results, this effect appears to be only partly reversed after a rapid return to 4 'C.…”

“…The fractions of initial stain intensities remaining at various dose levels of ultraviolet radiations were expressed using a semi-log plot. Of the 21 proteins which were identically resolved in the two electrophoretic systems used, 19 gave the same curves in both cases: L5, L6-7-7a, L8, L9, L11, L12, L13-13a, L14, L17-17a, L22, L26, L27-27a, L28, L29, L31, L32-33, L35a, L37, L38. In contrast, LIO, LlOa, L15, L18, L18a, L21, L23, L23a were determined using the pH-5.5 system (for the first dimension) while L3, L4, L25, L34-34a, L35, L36, L37a, L38a as also L19 and L37a were determined using the pH-8.6 system.…”

“…Preliminary experiments were done at 4 "C using 60-S subunits kept in a pH-7.4 buffer. Covalent protein-RNA cross-linking in subunits submitted to increasing doses of radiation was demonstrated with two methods already reported in a previous paper of this series concerning the photoinduced cross-linking of ribosomal proteins to the 18-S rRNA molecules in the intact 40-S subunits [6]. The first method consisted in the direct measurement of the amount of labelled ribosomal proteins which remained associated to RNA after addition of sodium dodecyl sulFate to the irradiated subunits.…”

“…It is worth noting that, among proteins of this group, proteins L3, L 5, L9, L 11, and L 36 were found to have lysine residues which became highly accessible to labelling in subunits heated from 4 "C to 25 "C [19]. Unshielded reactive groups might react unspecifically with RNA and consequently modify its secondary structure as inferred from the melting properties of irradiated subunits.…”