A study of the influence of magnesium ions on the conformation of ribosomal ribonucleic acid and on the stability of the larger subribosomal particle of rabbit reticulocytes.
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ABSTRACT:
SUBMITTER: Cox RA
PROVIDER: S-EPMC1164267 | biostudies-other | 1976 Dec
REPOSITORIES: biostudies-other
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Project description:The spectrum of RNA from the smaller and larger subparticles of rabbit reticulocyte ribosomes was studied as a function of pH, ionic strength, urea concentration and temperature. It was inferred that both RNA species form short double-helical segments of not more than about 10 base-pairs in length. Not more than about 70% of the base residues may be located in double-helical segments. RNA from the larger subparticle is richer in guanine and cytosine residues and its secondary structure is the more stable. These conclusions are based on the use of double-helical RNA from virus-like particles and of unfractionated Escherichia coli tRNA as model systems.
| S-EPMC1178836 | biostudies-other
Project description:1. The effect of removing Mg(2+) from a purified high-molecular-weight (1.07x10(6)) fraction of Escherichia coli ribosomal RNA was examined by ultracentrifugation, thermal denaturation and optical rotation. 2. At moderate I (0.1m-sodium chloride), EDTA at 2-50mm has little effect on RNA; at low I, 0.01-0.04 (with tris as counter-ion), two boundaries appear. 3. The leading boundary, S(20,w) about 20s, is identified with the original material with counter-ion Mg(2+) (;ionic atmosphere') removed, leading to an expanded form. 4. The slow boundary, 15-16s, is associated with a further loss of Mg(2+) and a further expansion, sensitive to EDTA concentration: it is proposed that this Mg(2+) is localized on the polynucleotide chain, i.e. ;site-bound'. 5. I is important and the EDTA effect at low I is reversible if Na(+) is added immediately after the EDTA: this Na(+) reversibility is lost on standing at 0 degrees . It is suggested that changes in the tertiary structure may be associated with this loss of reversibility. 6. Thermal-denaturation studies show that there is no loss of secondary structure associated with these changes: change in the optical-rotatory-dispersion spectrum in the region of the Cotton effect may be associated with this change in tertiary structure.
| S-EPMC1265099 | biostudies-other
Project description:The bacterial signal recognition particle (SRP) binds to ribosomes synthesizing inner membrane proteins and, by interaction with the SRP receptor, FtsY, targets them to the translocon at the membrane. Here we probe the conformation of SRP and SRP protein, Ffh, at different stages of targeting by measuring fluorescence resonance energy transfer (FRET) between fluorophores placed at various positions within SRP. Distances derived from FRET indicate that SRP binding to nontranslating ribosomes triggers a global conformational change of SRP that facilitates binding of the SRP receptor, FtsY. Binding of SRP to a signal-anchor sequence exposed on a ribosome-nascent chain complex (RNC) causes a further change of the SRP conformation, involving the flexible part of the Ffh(M) domain, which increases the affinity for FtsY of ribosome-bound SRP up to the affinity exhibited by the isolated NG domain of Ffh. This indicates that in the RNC-SRP complex the Ffh(NG) domain is fully exposed for binding FtsY to form the targeting complex. Binding of FtsY to the RNC-SRP complex results in a limited conformational change of SRP, which may initiate subsequent targeting steps.
| S-EPMC2612770 | biostudies-literature
Project description:A casein kinase was isolated and purifed from rabbit reticulocytes. About 90% of the enzyme activity co-sedimented with the ribosomal fraction, whereas about 10% of the enzyme activity was found in the ribosome-free supernatant. Both casein kinases (the ribosome-bound enzyme as well as the free enzyme) showed identical activity and the same molecular weight. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis a single band of about 70 000 mol.wt. was observed. Sucrose-gradient analysis, however, showed that the enzyme activity sedimented with a s20,w of approx. 7.5S. This observation suggested that the casein kinase is a dimer composed of subunits of identical molecular weight. The enzyme utilizes GTP as well as ATP as a phosphoryl donor. It preferentially phosphorylates acidic proteins, in particular the model substrates casein and phosvitin. Casein kinase is cyclic AMP-indepenoent. The Km values for ATP and GTP with phosvitin as a substrate were determined as 1.2 and 8.8 micrometer respectively.
| S-EPMC1164934 | biostudies-other
Project description:Mild proteolysis with Pronase selectively dissociates ribosomes not attached to mRNA into subunits; ribosomes attached to mRNA remain intact. A portion of monoribosomes from reticulocytes incubated with NaF resisted proteolytic dissociation. Recovery of mRNA from monoribosomes of NaF-treated reticulocytes therefore may be explained by persistent attachment of some monoribosomes to mRNA.
| S-EPMC1177715 | biostudies-other
Project description:Turnover of mRNA is a critical step that allows cells to control gene expression. Endoribonucleases, enzymes cleaving RNA molecules internally, are some of the key components of the degradation process. Here we provide a detailed characterization of novel endoribonuclease SLFN14 purified from rabbit reticulocyte lysate. Schlafen genes encode a family of proteins limited to mammals. Their cellular function is unknown or incompletely understood. In reticulocytes, SLFN14 is strongly overexpressed, represented exclusively by the short form, all tethered to ribosomes, and appears to be one of the major ribosome-associated proteins. SLFN14 binds to ribosomes and ribosomal subunits in the low part of the body and cleaves RNA but preferentially rRNA and ribosome-associated mRNA. This results in the degradation of ribosomal subunits. This process is strictly Mg(2+)- and Mn(2+)-dependent, NTP-independent, and sequence nonspecific. However, in other cell types, SLFN14 is a full-length solely nuclear protein, which lacks ribosomal binding and nuclease activities. Mutational analysis revealed the ribosomal binding site and the aspartate essential for the endonucleolytic activity of protein. Only few endoribonucleases participating in ribosome-mediated processes have been characterized to date. Moreover, none of them are shown to be directly associated with the ribosome. Therefore, our findings expand the general knowledge of endoribonucleases involved in mammalian translation control.
| S-EPMC4461289 | biostudies-literature
Project description:Most RNA folding studies have been performed under non-physiological conditions of high concentrations (≥10 mM) of Mg2+free, while actual cellular concentrations of Mg2+free are only ~1 mM in a background of greater than 50 mM Mg2+total. To uncover cellular behavior of RNA, we devised cytoplasm mimic systems that include biological concentrations of amino acids, which weakly chelate Mg2+. Amino acid-chelated Mg2+ (aaCM) of ~15 mM dramatically increases RNA folding and prevents RNA degradation. Furthermore, aaCM enhance self-cleavage of several different ribozymes, up to 100,000-fold at Mg2+free of just 0.5 mM, indirectly through RNA compaction. Other metabolites that weakly chelate magnesium offer similar beneficial effects, which implies chelated magnesium may enhance RNA function in the cell in the same way. Overall, these results indicate that the states of Mg2+ should not be limited to free and bound only, as weakly bound Mg2+ strongly promotes RNA function under cellular conditions.
| S-EPMC5984629 | biostudies-literature
Project description:Both monovalent cations and magnesium ions are well known to be essential for the folding and stability of large RNA molecules that form complex and compact structures. In the atomic structure of the large ribosomal subunit from Haloarcula marismortui, we have identified 116 magnesium ions and 88 monovalent cations bound principally to rRNA. Although the rRNA structures to which these metal ions bind are highly idiosyncratic, a few common principles have emerged from the identities of the specific functional groups that coordinate them. The nonbridging oxygen of a phosphate group is the most common inner shell ligand of Mg++, and Mg++ ions having one or two such inner shell ligands are very common. Nonbridging phosphate oxygens and the heteroatoms of nucleotide bases are common outer shell ligands for Mg++ ions. Monovalent cations usually interact with nucleotide bases and protein groups, although some interactions with nonbridging phosphate oxygens are found. The most common monovalent cation binding site is the major groove side of G-U wobble pairs. Both divalent and monovalent cations stabilize the tertiary structure of 23S rRNA by mediating interactions between its structural domains. Bound metal ions are particularly abundant in the region surrounding the peptidyl transferase center, where stabilizing cationic tails of ribosomal proteins are notably absent. This may point to the importance of metal ions for the stabilization of specific RNA structures in the evolutionary period prior to the appearance of proteins, and hence many of these metal ion binding sites may be conserved across all phylogenetic kingdoms.
| S-EPMC1370624 | biostudies-literature
Project description:1. RNA has been prepared from baby hamster kidney cells by extraction with a phenol-EDTA mixture and further purified by passing through a column of Sephadex G-25 that had been equilibrated with water. 2. Aging of the total RNA extracts at 4 degrees or heating at 95 degrees followed by rapid cooling caused a conversion of 28s RNA into material sedimenting in sucrose gradients at approx. 18s. 3. When heated RNA was re-extracted with phenol the sedimentation profile was not returned to that of the unheated RNA. 4. The 28s and 18s RNA fractions were collected separately from sucrose gradients by precipitation with 2vol. of ethanol and passed through a Sephadex G-25 column equilibrated with water. 5. Heat treatment of purified 28s RNA at 95 degrees caused the sedimentation coefficient to increase to approx. 40s, whereas similar treatment of 18s RNA caused no significant increase. If the RNA was heated before the Sephadex G-25 treatment the sedimentation coefficient of the 28s and 18s RNA decreased to approx. 12s and 8s. 6. Heating mixtures of purified 28s and 18s RNA at 95 degrees caused some aggregation of 18s material with the 28s fraction.
| S-EPMC1270179 | biostudies-other
Project description:Rat liver ribosomal RNA (rRNA) free from nuclease contaminants was isolated by a modification of the phenol technique. The 28s and 18s rRNA species were separated by preparative agar-gel electrophoresis. The two rRNA species were heated at different temperatures under various conditions and the amount of undegraded rRNA was determined by analytical agar-gel electrophoresis. The 18s rRNA remained unaltered after heating for up to 10min. at 90 degrees in water, acetate buffer, pH5.0, or phosphate buffer, pH7.0. Under similar or milder conditions 28s rRNA was partially degraded, giving rise to a well-delimited 6s peak and a heterogeneous material located in the zone between 28s and 6s. The dependence of degradation of 28s rRNA on the temperature and the ionic strength of the medium was studied. The greatest extent of degradation of 28s rRNA was observed on heating at 90 degrees in water. It is suggested that the instability of rat liver 28s rRNA is due to two factors: the presence of hidden breaks in the polymer chain and a higher susceptibility of some phosphodiester bonds to thermal hydrolysis.
| S-EPMC1185072 | biostudies-other