Effects of magnesium ions on ribosomes: A fluorescence study (original) (raw)
Related papers
Biochimica Et Biophysica Acta-general Subjects, 1997
The structural response of the ribosomes of the extremely thermophilic archaeon Sulfolobus solfataricus was analysed Ž . Ž . and compared to that of the mesophilic E. coli ribosomes by assaying ethidium bromide EB binding to the native 70S particles as a function of magnesium concentration. We found that the thermophilic ribosomes bound more EB than their mesophilic counterparts; on the other hand, inhibition of EB binding by Mg 2q ions was more effective in the E. coli 70S particle. In Sulfolobus, the separated 30S and 50S subunits and the 70S particle bound the drug in a similar fashion, whereas the E. coli 70S had a reduced number of binding sites with respect to the subunits. Light scattering measurements as a function of Mg 2q concentration were carried out at various temperatures to study the interaction between the ribosomal subunits from the thermophilic and the mesophilic bacteria. As expected, the association of ribosomal subunits in E. coli was magnesium dependent and could be observed also at low temperature. By contrast, the interaction between Sulfolobus ribosomal subunits was obligatorily dependent upon both magnesium ions and a temperature of at least 808C, close to the Ž . physiological optimum for cell growth 878C . Ž
Magnesium requirement for the formation of an active messenger RNA-ribosome-S-RNA complex
Journal of Molecular Biology, 1965
The interaction of single ribosomes with polyuridylic acid and S-RNA has been found to take place optimally in the presence of 15 rmr-magnesium ions. In contrast, one-half as much magnesium ions is required for amino acid poly. m erization by the product of this interaction, or by naturally occurring polyribosomes. These obs ervations are believed to account for the higher magnesium ion requirement of amino acid inc orporation stimulated by added polynucleotide, as compared with that needed for the endogenous activity of systems to which no m essenger RNA is added.
Journal of Molecular Biology, 1995
In previous studies we have shown that specific nuclear pre-mRNAs and 1 Department of Genetics their splicing products, as well as the general population of nuclear poly(A) + The Hebrew University of RNA, are found packaged in 200 S large nuclear ribonucleoprotein (lnRNP) Jerusalem, Jerusalem 91904, particles that represent the splicing machinery in vivo. The lnRNP particles Israel contain all U small nuclear ribonucleoproteins (snRNPs) required for 2 Department of Organic splicing, as well as several proteins including non-snRNP splicing factors. Chemistry, The Weizmann Here we show that upon addition of EDTA to sucrose gradient-fractionated Institute of Science Rehovot 200 S particles, part of their components (e.g. part of the U snRNPs) are no 76100, Israel longer associated with pre-mRNAs, which are now packaged in 70 S particles. This 200 S to 70 S transition makes the pre-mRNA more susceptible to digestion by RNase. The effect of EDTA is reversible, as back addition of Mg 2+ results in the reconstitution into 200 S lnRNP particles of: (1) all five snRNPs required for splicing; (2) the SR proteins; and (3) CAD mRNA, as a representative of nuclear RNA polymerase II transcripts. Remarkably, electron microscopy of the reconstituted particles shows a compact structure, 50 nm in diameter, that is indistinguishable from the original undissociated particles. We conclude that Mg 2+ is required for the integrity of the 200 S lnRNP particles.
Effects of magnesium ions on the stabilization of RNA oligomers of defined structures
RNA, 2002
Optical melting was used to determine the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concentration. The oligomers included helices composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concentration on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temperature or free energy) was three times greater than that observed for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes observed upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes observed in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the observed cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calculated.
Biochemistry, 1998
The effects of Mg 2+ concentration, subunit association, and temperature on the structure of 16S rRNA in the Escherichia coli ribosome were investigated using UV cross-linking and gel electrophoresis analysis. Mg 2+ concentrations between 1 and 20 mM and temperatures between 5 and 55°C had little effect on the frequency of 12 of the 14 cross-links in 30S subunits and modest effects on the same cross-links in 70S ribosomes. In contrast, two cross-links, C967 × C1400 and C1402 × C1501, involving rRNA in the decoding region are present in 30S subunits only above 3 mM Mg 2+ , increase in frequency at higher Mg 2+ concentration, and are both more frequent when 50S subunits are included in the reactions. In 70S ribosomes, the cross-link C1402 × C1501 increases but the cross-link C967 × C1400 decreases at higher Mg 2+ concentrations. One cross-link, C1397 × U1495, is detected only in 70S ribosomes and decreases in frequency as Mg 2+ concentration is increased. An additional cross-link, A1093 × C1182, decreases upon subunit association. The cross-link frequency differences indicate that the arrangement of the decoding region of the 16S rRNA, but not in the rest of the subunit, is readily altered by Mg 2+ ions and subunit association.
The Dissociation of Escherichia coli Ribosomes
European Journal of Biochemistry, 1973
A systematic study has been made of the dissociation into subunits of the ribosomes of Escherichia coli. By analytical ultracentrifugation, ribosomes were shown to be in dynamic reversible equilibrium with their subunits. This enabled a thermodynamic analysis to be made of the association-dissociation reaction and of several factors which influence it : the concentration of monovalent and divalent cations, the presence of transfer RNA and supernatant factors, the pH, and hydrostatic pressure. The apparent change in free energy for the association in 10 mM sodium phosphate pH 7.0 was-17.4 kcal/mol. Association was accompanied by tthe net binding of mabqesium ions or the net release of monovalent cations (potassium, sodium, or Tris ions). Circular dichroism measurements of ribosomes and their isolated subunits over a range of magnesium and potassium ion concentrations indicated that a change in the conformation of each subunit accompanied association. The change occurring in the 50-S subunit was dependent on the presence of potassium ions, whereas the change in the 30-S subunit was magnesium-ion dependent. The conformational change in the 60-S subunit was critical for the association-dissociation reaction. Under ionic conditions which lead to ribosome dissociation one molecule of transfer RNA was released per 70-S ribosome. This was an independent reaction which did not influence the subunit association-dissociation equilibrium. The release of transfer RNA was accompanied by the net release of four magnesium ions, and occurred with an apparent change in the free energy of 20.8 kcal/mol .
Biochemistry, 1986
The imino proton nuclear magnetic resonance spectrum of Escherichia coli 5s ribonucleic acid (RNA) changes when the Mg2+ ion concentration drops below physiological levels. The transition between the physiological and low magnesium spectral forms of 5s R N A has a midpoint a t approximately 0.3 m M Mg2+. Many of the most conspicuous changes observed in the downfield spectrum of 5s R N A as the magnesium concentration is reduced are due to adjustments in the structures of helices I and IV and the disappearance of resonances originating in helix V. The binding of ribosomal protein L25 to 5s R N A in the absence of magnesium stabilizes helix V structures.
Journal of Biological Chemistry, 1996
The effects of magnesium ions on a 32-mer ribozyme (R32) were examined by high resolution NMR spectroscopy. In solution, R32 (without its substrate) consisted of a GAAA loop, stem II, a non-Watson-Crick 3-base pair duplex and a 4-base pair duplex that included a wobble G:U base pair. When an uncleavable substrate RNA (RdC11) was added to R32 without Mg 2؉ ions, a complex did not form between R32 and RdC11 because the substrate recognition regions of R32 formed intramolecular base pairs (the recognition arms were closed). By contrast, in the presence of Mg 2؉ ions, the R32-RdC11 complex was formed. Moreover, titration of mixtures of R32 and RdC11 with Mg 2؉ ions also induced the ribozymesubstrate interaction. Elevated concentrations (1.0 M) of monovalent Na ؉ ions could not induce the formation of the R32-RdC11 complex. These data suggest that Mg 2؉ ions are not only important as the true catalysts in the function of ribozyme-type metalloenzymes, but they also induce the structural change in the R32 hammerhead ribozyme that is necessary for establishment of the active form of the ribozyme-substrate complex. 1 The abbreviations used are: R32, 32-mer ribozyme; HPLC, high pressure liquid chromatography; NOE, nuclear Overhauser effect.
Modification of the accessibility of ribosomal proteins after
1990
Free- and EF-2-bound 80 S ribosomes, within the high-affinity complex with the non-hydrolysable GTP analog: guanylylmethylenediphosphonate (GuoPP(CH2)P), and the low-affinity complex with GDP, were treated with trypsin under conditions that modified neither their protein synthesis ability nor their sedimentation constant nor the bound EF-2 itself. Proteins extracted from trypsin-digested ribosomes were unambiguously identified using three different two-dimensional gel electrophoresis systems and 5 S RNA release was checked by submitting directly free- and EF-2-bound 80 S ribosomes, incubated with trypsin, to two-dimensional gel electrophoresis. Our results indicate that the binding of (EF-2)-GuoPP[CH2]P to 80 S ribosomes modified the behavior of a cluster of five proteins which were trypsin-resistant within free 80 S ribosomes and trypsin-sensitive within the high-affinity complex (proteins: L3, L10, L13a, L26, L27a). As for the binding of (EF-2)-GDP to 80 S ribosomes, it induced an intermediate conformational change of ribosomes, unshielding only protein L13a and L27a. Quantitative release of free intact 5 S RNA which occurred in the first case but not in the second one, should be related to the trypsinolysis of protein(s) L3 and/or L10 and/or L26. Results were discussed in relation to structural and functional data available on the ribosomal proteins we found to be modified by EF-2 binding.