Melting of local ordered structures in yeast 5S ribosomal RNA in aqueous salts.

Equilibrium and kinetics of thermal melting of yeast 5S ribosomal RNA in aqueous NaCl with or without Mg2+ were investigated by differential thermal melting and temperature jump methods. Two peaks (1 and 2) and a shoulder were observed in each of melting curves at ionic strength I=0.002-0.5 and linearity between each of melting temperatures T1m and T2m and log I was found at I=0.01-0.5 in the Mg2+-free solution. The local structures were found to be stabilized considerably by Mg2+. The temperature jump measurements gave the kinetic melting curve of the structure 1 at I=0.03 without Mg2+ or with 0.5 mM Mg2+. The kinetic Tm coincided well with the corresponding static Tm. For the structure 1, various parameters were calculated from the kinetic data, which indicated a double helical character of the structure 1. In terms of the values of Tm, G-C content, and enthalpy change of the transition of the structure 1 or 2, appropriateness of each of the secondary structure models of eukaryotic 5S RNA proposed previously was discussed.     

Base pairing in wheat germ ribosomal 5S RNA as measured by ultraviolet absorption, circular dichroism, and Fourier-transform infrared spectrometry

Ultraviolet absorption (UV) and circular dichroism (CD) spectra of wheat germ 5S RNA, when compared to tRNAPhe, indicate a largely base-paired and base-stacked helical structure, containing up to 36 base pairs. Fourier-transform infrared (FT-IR) spectra of tRNAPhe and wheat germ ribosomal 5S RNA have been acquired at 30 and 90 degrees C. From the difference of the FT-IR spectra between 90 and 30 degrees C, the number of base pairs in both RNAs was determined by modification of a previously published procedure [Burkey, K. O., Marshall, A. G., & Alben, J. O. (1983) Biochemistry 22, 4223-4229]. The base-pair composition and total base-pair number from FT-IR data are now consistent for the first time with optical (UV, CD, Raman) and NMR results for ribosomal 5S RNA. Without added Mg2+, tRNAPhe gave 18 +/- 2 base pairs [7 A-U and 11 G-C], in good agreement with the number of secondary base pairs from X-ray crystallography [8 A-U, 12 G-C, and 1 G-U]. Within the 10% precision of the FT-IR method, wheat germ 5S RNA exhibits essentially the same number of base pairs [14 A-U, 17 G-C, and 5 G-U; for a total of 36] in the absence of Mg2+ as in the presence of Mg2+ [14 A-U, 18 G-C, and 3 G-U; for a total of 35], in agreement with the UV hyperchromism estimate of G-C/(A-U + G-C) = 0.58.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational comparisons among homologous bony fish 5S ribosomal RNAs

The reversible thermal melting of homologous 5S ribosomal RNAs of three bony fishes, rainbow trout (Salmo gairdneri), carp (Cyprinus carpio), and dog salmon (Onchorhynchus keta), were compared in aqueous salts (sodium chloride and/or divalent metal chlorides) by means of differential optical absorption at 260 and 280 nm, and c.d. at 265 nm. The melting parameters of the multiphasic melting process were conserved among the homologous RNAs, and it was shown that they did not depend on the methods of measurement but on concentrations of [Na+] and of [metal2+]. Other physicochemical methods, such as FT-IR absorption, gave the same melting profiles as the u.v. methods. The c.d. spectrum was homologous among the ribosomal RNAs. The melting is explained by changes in local structures in the 'consensus secondary structure model'. Also, stabilization of the secondary and tertiary structures by binding of Mg2+ and Ca2+, and the catalysed hydrolysis of the main chains by various divalent ions are discussed.     

Base pairing in Bacillus subtilis ribosomal 5S RNA as measured by ultraviolet absorption and Fourier-transform infrared spectrometry

Ultraviolet (260 and 280 nm) and Fourier-transform infrared (FT-IR) spectra of Bacillus subtilis ribosomal 5S RNA have been acquired between 20 and 90 degrees C. In the presence of added Mg2+, the average UV melting midpoint, Tm, is 60 (A260) or 62 degrees C (A280), resolving into two components (Tm = 54 and 68 degrees C). In the presence of 10 mM Mg2+, the normalized A260 increases by about 5%, and the average Tm increases to 70 degrees C (A260 or A280), resolving into components at 63 and 73 degrees C at 260 nm but not resolved at 280 nm. From the difference of the 5S RNA FT-IR spectra between 90 and 30 degrees C, the number of base pairs in B. subtilis 5S RNA was determined by the procedure outlined in the accompanying paper [Li, S.-J., Burkey, K. O., Luoma, G. A., Alben, J. O., & Marshall, A. G. (1984) Biochemistry (preceding paper in this issue)]. Addition of 10 mM Mg2+ increases the number of A-U pairs by 1 (from 11 to 12) and the number of G-C pairs by 2 (from 15 to 17). FT-IR melting curve midpoints show that addition of Mg2+ increases the melting point for both A-U and G-C pairs in B. subtilis 5S RNA. The A-U pairs melt before G-C pairs (56 vs. 64 degrees C) in the absence of Mg2+, but both types of pairs melt at the same temperature (67 vs. 70 degrees C) in the presence of Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of base pairing in ribonucleic acid by Fourier-transform infrared spectrometry: yeast ribosomal 5S ribonucleic acid

Fourier-transform infrared (FT-IR) spectra of yeast ribosomal 5S RNA have been acquired at several temperatures between 30 and 90 degrees C. The difference spectrum between 90 (bases unstacked) and 30 degrees C (bases stacked) provides a measure of base stacking in the RNA. Calibration difference spectra corresponding to stacking of G-C or A-U pairs are obtained from "reference" FT-IR spectra of poly(rG) X poly(rC) minus 5'-GMP and 5'-CMP or poly(rA) X poly(rU) minus 5'-AMP and 5'-UMP. The best fit linear combination of the calibration G-C and A-U difference spectra to the 5S RNA (90-30 degrees C) difference spectrum leads to a total of 25 +/- 3 base pairs (17 G-C pairs + 8 A-U pairs) for the native yeast 5S RNA in the absence of Mg2+. In the presence of Mg2+, an additional six base pairs are detected by FT-IR (one G-C and five A-U). FT-IR melting curve midpoints show that A-U and G-C pairs melt together (65 and 63 degrees C) in the presence of Mg2+ but A-U pairs melt before G-C pairs (47 vs. 54 degrees C) in the absence of Mg2+.     

Multistage unfolding of wheat germ ribosomal 5S RNA analyzed by differential scanning calorimetry

Unfolding of purified wheat germ ribosomal RNA has been studied by differential scanning calorimetry (DSC) from 15 to 95 degrees C, in the presence and absence of 100 mM NaCl and/or 10 mM MgCl2. The total enthalpy of melting varies from about 290 (no sodium or magnesium) to 480 kcal/mol (Mg2+ only) and precisely accounts for the number and types of base pairs deduced from prior Fourier-transform infrared experiments. The composite DSC curves are analyzed into four individual two-state melting stages. Both Na+ and Mg2+ shift the melting transitions to higher temperature; in addition, Mg2+ causes the individual transitions to merge (i.e., melt cooperatively) and leads to irreversible chain cleavage at high temperature. The results are analyzed according to three general secondary base-pairing models for eukaryotic 5S RNA.     

The role of magnesium and potassium ions in the molecular mechanism of ribosome assembly: hydrodynamic, conformational, and thermal stability studies of 16 S RNA from Escherichia coli ribosomes

In an attempt to understand the role of magnesium ion in ribosome assembly in vitro, the hydrodynamic shape, conformation, and thermal stability of ribosomal 16 S RNA were studied systematically as a function of Mg2+ concentration by sedimentation velocity, intrinsic viscosity, circular dichroism, and difference ultraviolet absorption spectroscopy. These results were then compared with the corresponding parameters obtained for 16 S RNA under the optimal conditions of reconstitution, i.e., at 37 degrees C, 20 mM Mg2+, an ionic strength equal to 0.37, and pH 7.8 [S. H. Allen, and K.-P. Wong (1978) J. Biol. Chem. 253, 8759-8766]. When the 360 mM KCl required for reconstitution of 30 S ribosomes is added to the medium, only subtle conformational changes are observed, consistent with the destabilization of the conformation, thus making the RNA molecule more "open" and accessible to protein binding. However, when the concentration of Mg2+ is lowered from 20 to 1 mM, the hydrodynamic parameters indicate that the 16 S RNA is partially unfolded, while thermal denaturation studies suggest that the amount of base-stacking and base-pairing is not concomitantly altered. Further removal of the Mg2+ by dialysis against a pH 7.8 buffer containing no Mg2+ results in a drastic decrease of secondary structure and indicates that the Mg2+ is required for maintenance of the pairing, stacking, and stability of the nucleotide bases, in addition to the long range interactions which result in a compact structure. The results suggest that the 20 mM Mg2+ is required for the 16 S RNA molecules to assume the proper secondary and tertiary structure containing the protein-binding sites, while the high K+ concentration (360 mM KCl) is needed for "loosening up" the RNA, making the protein binding sites more accessible to the ribosomal proteins for molecular recognition and binding as well as for the conformational changes that occur during ribosome assembly.     

Effect of magnesium ion on the structure of the 5S RNA from Escherichia coli. An imino proton magnetic resonance study of the helix I, IV, and V regions of the molecule

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 RNA has a midpoint at approximately 0.3 mM Mg2+. Many of the most conspicuous changes observed in the downfield spectrum of 5S RNA 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 RNA in the absence of magnesium stabilizes helix V structures.     

Physical properties of ribosomal ribonucleic acid isolated from bacteria deficient in ribonuclease I

1. Bacteria deficient in ribonuclease I were used as a source of stable ribosomal RNA. RNA was isolated from a ribosome fraction of Pseudomonas fluorescens N.C.I.B. 8248 and acetone-treated cells of Escherichia coli M.R.E. 600 by the method developed by Robinson & Wade (1968). 2. The s(20,w) of the 16S and 23S components can vary from 21S and 28S down to 4S depending on the RNA macro-ion concentration and the extent to which charge is suppressed by univalent Na(+) and tris(+) counter-ions or neutralized through the binding of bivalent Mg(2+) to phosphate groups. 3. The primary charge effect in sedimentation and the frictional coefficient (which increases as the molecular conformation expands) both increase with charge and cause a decrease in s value. 4. RNA solutions heated to 80 degrees C for 10min show minor changes in s value and a detectable increase in polydispersity. Millimolar concentrations of Mg(2+) promote heat-instability and so does treatment of RNA solutions with the nuclease adsorbent macaloid, which was found to contaminate the solutions with Mg(2+). 5. The stabilization of secondary structure by univalent and bivalent cations was investigated by optical methods. 6. The sedimentation properties of 30S and 50S ribosomal subunits and their constituent 16S and 23S RNA components were compared and discussed from the viewpoint of unfolding.     

Changes in the conformation and stability of 5 S RNA upon the binding of ribosomal proteins.

The binding of ribosomal proteins L25, L18, and L5 to 5 S RNA results in a conformational change and a destabilization of the 5 S RNA molecule. The changes observed in the near ultraviolet circular dichroism (CD) spectra and in the melting profiles indicate an increase in base stacking uith an accompanying increase in asymmetry of the bases and a decrease in the conformational stability of the 5 S RNA. These results are consistent with the interpretation that the binding of these proteins increases the stacking of specific single-stranded bases in 5 S RNA and aligns them in helical arrays, resulting in a conformation which facilitates base-pairing with nucleotide segment(s) of the ribosomal 23 S RNA or the transfer RNA (or both). The simple and precise difference CD method described here is potentially useful for studying subtle conformational changes of other nucleic acid-protein interactions.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45 degrees C in the absence and presence of Mg2+. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. delta H0 and delta S0 for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, delta H0 approximately 15-16 kcal/mol (63-67 kJ/mol) and delta S0 approximately 50-57 cal/K per mol (209-239 J/K per mol). The presence of Mg2+ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg2+, the delta H0 and delta S0 values are larger and temperature-dependent, with delta H0 approximately 22 kcal/mol (92 kJ/mol) and delta S0 approximately 72 cal/K per mol (approx. 300 J/K per mol) at 25 degrees C, and delta Cp0 approximately 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

Partial melting of the segment around pseudouridine in yeast 5S RNA

Pseudouridine in yeast 5S RNA was modified with 4-bromomethyl-7-methoxy-coumarin(BMC). Temperature dependence of fluorescence intensity was measured at various concentrations of Mg2+ and K+ cations. Hyperchromicity was also measured. At 100mM KCl and 10mM Mg2+, fluorescence intensity decreased with temperature as free BMC except a plateau at 45 degrees C. Withdrawal of Mg2+ from the buffer resulted in a large quenching at 20 degrees C and showed a gradual increase of fluorescence intensity with temperature, indicating a partial melting of the segment around pseudouridine. The temperature range agrees with the low melting temperature shown by hyperchromicity. In 10 mM KCl solution, the effects are more exaggerated.     

Conserved unpaired adenine residues are important for ordered structures of 5S ribosomal RNA. An infrared study of the secondary and tertiary structure of Thermus thermophilus 5S rRNA

An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+. These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups. T. thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure. Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs. Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA.     

Studies on the negative circular dichroic bands around 297 nm of ribosomes from bacterial cells.

The small negative CD bands around 297 nm of isolated 30-S and 50-S ribosomal subunits were precisely measured for three bacteria, Bacillus stearothermophilus, Bacillus subtilis and Escherichia coli Q 13. The intensities of the negative CD bands of 30-S subunits were always much greater than those of 50-S subunits irrespective of the bacterial strains, which may be related to the difference in comformations of rRNAs and proteins in the complexes between these subribosomal particles. The dissociation of 70-S ribosomes into two subunits by lowering Mg2+ concentration caused evident enhancement of intensity of the 297 nm CD band, which was completely reversed by the association of the two subunits into 70-S particles. The melting profiles of CD spectra 3 B. stearothermophilus and E. coli were compared and both subunits of the former were found to be more heat stable than those of the latter. It was found that 5 M urea and 0.5% sodium dodecyl sulfate (SDS) treatment caused considerable reduction of the negative CD intensity around 297 nm of 30-S subunits but no significant change of 50-S subunits, while no significant change was observed for the CD spectra of isolated 16-S and 23-S rRNAs by the same treatment. Effects of EDTA treatment and then addition of Mg2+ on the CD spectra and fluorescence emission spectra of the subunits were also observed and the contribution by the interaction between rRNA s and proteins in ribosomes to the small negative band around 297 nm was discussed.     

Solution structure and thermodynamics of a divalent metal ion binding site in an RNA pseudoknot.

Identification and characterization of a metal ion binding site in an RNA pseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)63+, as a probe for magnesium (II) hexahydrate, Mg(H2O)62+, in nuclear magnetic resonance (NMR) structural studies. The pseudoknot causes efficient -1 ribosomal frameshifting in mouse mammary tumor virus. Divalent metal ions, such as Mg2+, are critical for RNA structure and function; Mg2+preferentially stabilizes the pseudoknot relative to its constituent hairpins. The use of Co(NH3)63+as a substitute for Mg2+was investigated by ultraviolet absorbance melting curves, NMR titrations of the imino protons, and analysis of NMR spectra in the presence of Mg2+or Co (NH3)63+. The structure of the pseudoknot-Co(NH3)63+complex reveals an ion-binding pocket formed by a short, two-nucleotide loop and the major groove of a stem. Co(NH3)63+stabilizes the sharp loop-to-stem turn and reduces the electrostatic repulsion of the phosphates in three proximal strands. Hydrogen bonds are identified between the Co(NH3)63+protons and non-bridging phosphate oxygen atoms, 2' hydroxyl groups, and nitrogen and oxygen acceptors on the bases. The binding site is significantly different from that previously characterized in the major groove surface of tandem G.U base-pairs, but is similar to those observed in crystal structures of a fragment of the 5 S rRNA and the P5c helix of the Tetrahymena thermophila group I intron. Changes in chemical shifts occurred at the same pseudoknot protons on addition of Mg2+as on addition of Co(NH3)63+, indicating that both ions bind at the same site. Ion binding dissociation constants of approximately 0.6 mM and 5 mM (in 200 mM Na+and a temperature of 15 degrees C) were obtained for Co(NH3)63+and Mg2+, respectively, from the change in chemical shift as a function of metal ion concentration. An extensive array of non-sequence-specific hydrogen bond acceptors coupled with conserved structural elements within the binding pocket suggest a general mode of divalent metal ion stabilization of this type of frameshifter pseudoknot. These results provide new thermodynamic and structural insights into the role divalent metal ions play in stabilizing RNA tertiary structural motifs such as pseudoknots.     

The 3'' terminus of 16S rRNA: secondary structure and interaction with ribosomal protein S1.

We report studies of the secondary structure and S1 ribosomal protein binding properties of the colicin fragment, containing 49 residues from the 3' terminus of E. coli 16S rRNA. Temperature jump relaxation kinetic measurements reveal two helices in the structure. One of these, melting at 81 degrees C in 5 mM Mg2+, is associated with the 9-base pair hairpin helix predicted by the nucleotide sequence. The other melting transition, at 21 degrees C in 5 mM Mg2+, is assigned to a 4-base pair helix which constrains the pyrimidine tract of the colicin fragment into a bulge loop. S1 protein forms a strong 1:1 complex with the colicin fragment, with an association constant of 5 x 10(6) M-1 in 5 mM Mg2+. More protein molecules are bound, but with weaker affinity, when the S1 concentration is increased. S1 binding causes melting of the colicin fragment secondary structure, as inferred from the observed absorbance increase. The S1 binding site on the colicin fragment has been localized in the region of the bulge loop, since the melting transition corresponding to the 4-base pair helix is lost in the complex. We discuss current models for the role of S1 protein in polypeptide chain initiation in light of these and previous results.     

The effects of magnesium ions on the hydrodynamic shape,conformation, and stability of the ribosomal 23S RNA from E.coli

The hydrodynamic shape, conformation, and thermal stability of ribosomal 23S RNA have been studied by sedimentation velocity analysis, circular dichroism, ultraviolet absorption, and thermal denaturation. The results show that magnesium ion is critically required for maintaining the structural integrity of ribosomal 23S RNA. Upon removal of magnesium ions, the sedimentation coefficient of 21.1 S for native 23 S RNA is reduced to approximately 8.5 S, indicative of a large unfolding of the RNA. A large increase in absorbance at 260 nm in UV spectra of 23S RNA and a decrease in ellipticity near the 265 nm peak which was followed by parallel changes in the 237 nm trough and the 208 nm trough. Furthermore, the melting temperature (T_m) of the 23S RNA is lowered to 50°C from 65°C with a concomitant decrease in the cooperativity of the melting curve of 23S RNA.     

Structural and functional exchangeability of 5 S RNA species from the eubacterium E.coli and the thermoacidophilic archaebacterium Sulfolobus solfataricus.

The role of 5 S RNA within the large ribosomal subunit of the extremely thermophilic archaebacterium Sulfolobus solfataricus has been analysed by means of in vitro reconstitution procedures. It is shown that Sulfolobus 50 S subunits reconstituted in the absence of 5 S RNA are inactive in protein synthesis and lack 2-3 ribosomal proteins. Furthermore, it has been determined that in the course of the in vitro assembly process Sulfolobus 5 S RNA can be replaced by the correspondent RNA species of E.coli; Sulfolobus reconstituted particles containing the eubacterial 5 S molecule are stable and active in polypeptide synthesis at high temperatures.