Pnina Spitnik-Elson

Detachment of ribosomal proteins by salt: I. Effect of conditions on the amount of protein detached

Abstract The amount of protein detached from ribosomes by salt varies markedly with the conditions used, increasing with rising salt concentration and decreasing with rising Mg 2+ or ribosome concentration. The detached proteins are different from those remaining attached to the RNA. In the presence of Mg 2+ , where the ribosomes maintain a compact structure, the release of protein is slow and different salts have different effectivities, NaCl detaching large amounts of protein and NH 4 Cl none. In the absence of Mg 2+ , where the ribosomes are unfolded, the release is rapid and the differences among salts tend to disappear; NH 4 Cl is nearly as effective as NaCl. The dependence on salt and ribosome concentration remains, but the amount of protein detached from unfolded ribosomes is much greater, reaching 96% in 2 m -LiCl. Thus, nearly any desired amount of ribosomal protein can be detached by suitable manipulation of the experimental conditions. It is concluded that the conformation of the ribosome is an important factor in determining the detachment of ribosomal proteins by salt. It is also concluded that electrostatic forces have a major role in binding virtually all of the ribosomal proteins to the ribosomal RNA, and that different proteins have different affinities for the RNA.

Detachment of ribosomal proteins by salt: II. Some properties of protein-deficient particles formed by the detachment of ribosomal proteins

Abstract We have examined certain properties of protein-deficient particles produced by detaching varying amounts of protein from Escherichia coli ribosomes with 0.5 m -NaCl, 1 m -NaCl or 2 m -LiCl. The particles are highly vulnerable to ribonuclease, and their sedimentation coefficients are very sensitive to changes in the ionic environment and in particle concentration. This behaviour does not seem qualitatively different from that reported for unfolded ribosomes from which no protein was detached, and would appear to be due more to the opening up of the ribosomal structure than to the removal of proteins per se . The removal of relatively small amounts of protein from ribosomes causes marked changes in the sedimentation coefficients; but with the detachment of less than half of the original protein, the coefficients approach a lower limiting value and become virtually insensitive to the further removal of protein, giving particles of different protein content which have the same sedimentation coefficient. The limiting value for particles formed in 0.5 m -NaCl is different from that for particles formed by treatment with 1 m -NaCl or 2 m -LiCl, making it possible in this case to form particles with the same protein content but different sedimentation coefficients. It is concluded that the sedimentation coefficient is not a reliable indicator of the amount or kind of proteins present in particles, and cannot be used as the sole basis for characterizing particles or comparing different particles. Biologically active 30 s ribosomes have been reassembled from inactive NaCl and LiCl particles and the proteins detached from them.

The solubilization of ribosomal protein from Escherichia coli

Abstract The protein of Escherichia coli ribosomes has been prepared by allowingthe ribosomal RNAase to digest the ribosomal RNA under dialysis against various salts and buffers. The solubility of the protein was found to depend on the kind of salt present and on the pH, ionic strength, and temperature of the medium. At neutral pHs the solubility increased with increasing ionic strength, and complete solubilization of up to 17 mg/ml of ribosomal protein was attained in 1 M Tris buffer (pH 7.4).

The effect of trypsin on 30-S and 50-S ribosomal subunits of Escherichia coli

Abstract The effect of soluble trypsin on ribosomal subunits of Escherichia coli has been studied by analyzing the ribosomal proteins in a two-dimensional acrylamide gel electrophoresis system. In order to correlate the susceptibility of the ribosomal proteins to trypsin with the structure of the ribosomes, folded and unfolded 30-S and 50-S subunits were tested. The results reported here indicate that all the ribosomal proteins were susceptible to trypsin in the compact as well as in the unfolded ribosome; however, some proteins were less susceptible than others. When the ribosome was unfolded, tryptic digestion was faster, but there was little indication of significant changes in the relative vulnerabilities of the different proteins. The relative resistance of some of the proteins may therefore probably be attributed to protection of the specific sites of tryptic attack rather than to the location of the protein in the ribosomal structure.

A new approach to the fractionation of ribosomal protein.

Earlier work [l] in this laboratory has shown that about 95% of the ribosomal proteins can be detached from the ribosomal RNA with salt, provided that the ribosomes are unfolded before being treated with the salt. The amount of protein detached from such unfolded ribosomes is a function of the salt concentration, and at a fixed salt concentration it is a function of the ribosome concentration. We have proposed that the dependence on ribosome concentration may result from an equilibrium between the detachment and reattachment of proteins. In this case, if the detached proteins were to be continuously removed from the ribosomes, it might be possible to obtain a small group of ribosomal proteins whose detachment would depend only on the salt concentration employed, and it might then be possible to isolate different groups of proteins by employing successively different salt concentration. We have tried to do this by attaching unfolded 50 S ribosomal subunits to a fixed support and washing them with a salt gradient. The preliminary results, reported here, indicate that the ribosomal proteins can be fractionated in this way. As the fvted support, we employed a column of DEAE-cellulose. Native folded ribosomes can be eluted from such a column with salt at neutral pH [2] However, adsorbed ribosomal RNA cannot be eluted [3] until the ion exchanger is deionized with alkali. Since unfolded ribosomes are essentially strands of RNA partly neutralized by the attached proteins, it seemed likely that their chromatographic behaviour would resemble that of ribosomal RNA, and this proved to be the case. Therefore, unfolded 50 S ribosomal subunits were adsorbed on a DEAE-

Fractionation of ribosomal protein from Escherichia coli by ammonium sulfate precipitation.

A solution of the total ribosomal protein of E. coli has been separated into three fractions by ammonium sulfate precipitation. The solubility and electrophoretic behaviour of the fractions were examined at different pHs at ionic strength 0.02. The fractions differed from each other in both respects. It has been shown that the ribosomal protein contains proteins with widely different isoelectric points, which can interact with one another at low ionic strength to form insoluble complexes.

Long range RNA-RNA interactions in the 30 S ribosomal subunit of E. coli.

We have attempted to identify long-range interactions in the tertiary structure of RNA in the E. coli 30 S ribosome. Native subunits were cleaved with ribonuclease and separated into nucleoprotein fragments which were deproteinized and fractionated into multi-oligonucleotide complexes under conditions intended to preserve RNA-RNA interactions. The final products were denatured by urea and heat and their constituent oligonucleotides resolved and sequenced. Many complexes contained complementary sequences known to be bound together in the RNA secondary structure, attesting to the validity of the technique. Other co-migrating oligonucleotides, not joined in the secondary structure, contained mutually complementary sequences in locations that allow base-pairing interaction without disrupting pre-existing secondary structure. In seven instances the complementary relationship was found to have been preserved during phylogenetic diversification.

The detachment of ribosomal proteins by urea: evidence for non-electrostatic RNA-protein interaction in the ribosome

Native ribosomes may be adsorbed on a DEAEcellulose column and eluted with salt without distroying their structure or biological activity [l] . We have shown, however, that if the ribosomes are first unfolded by magnesium depletion at low ionic strength [2] and are then adsorbed on DEAE-cellulose, the ribosomal proteins can be detached without dissociating the RNA from the column [3]. Under these conditions, where the negative potential of the RNA is largely neutralized by the positively charged groups of the ion exchanger, proteins were detached at ionic strengths much lower than those required when the unfolded ribosomes are free in solution [4]. We also observed that 6 M urea detached a large part of the ribosomal protein at a salt concentration (1 mM sodium phosphate) which removed no protein in the absence of urea. These observations were made with 50 S ribosomal subunits [31. The experiments reported in the present communication were performed to obtain information on two points: (a) the behavior of the 30 S ribosomal subunit under similar conditions and (b) the identity of the proteins in the two groups, those detached by urea and those not detached by urea alone. For this purpose unfolded 30 S subunits in 6 M urea 1 mM sodium phosphate were applied to a DEAEcellulose column equilibrated with the same solvent. Under these conditions the unfolded ribosomes were completely adsorbed, but 34% of the proteins came

Studies on the ribosome and its components.

Publisher Summary This chapter describes that the bacterial ribosome is a noncovalent complex made up of three molecules of RNA, over fifty different protein molecules, and certain monovalent and bivalent cations, arranged in two interacting but separable subunits. The ribosome is the site of protein synthesis and carries at least two catalytic sites that participate in this process, and serves as the focus and organizing center of the protein-synthesizing apparatus. As such, it interacts with other macromolecular components of the apparatus: messenger RNA, aminoacyl- and peptidyl-tRNAs, and the numerous protein factors that assist in peptide chain initiation, elongation and termination. Each of these ligands interacts with a specific binding site on the surface of the ribosome in a strictly controlled sequence: binding; acting on or being acted on by the ribosome; in some cases, being transferred to a different site; and eventually becoming detached. The chapter discusses the experiments having to do with the nature of the ribosomal proteins, protein-RNA interactions, conformation and conformational changes, and the specific cations required for the structural integrity of the ribosome.

Aggregation properties of ribosomal protein from Escherichia coli

Abstract Treatment with acidic ethanol accomplishes the separation of the ribosomal proteins into two main fractions, one soluble in 0.25 M HCl-80% ethanol and one insoluble. Further purification of the crude fractions by titration gave two basic fractions which, while still heterogeneous, were soluble in aqueous solution over almost the entire pH range. The amino acid composition and electrophoretic behavior of the fractions are described. Different aggregation procedures for the whole ribosomal protein are discussed.