IA. Cause and Effect . . . . . . . . . . . . . . 465 1.2. Prerequisites of Selforganization . . . . . . . 467 1.2.3. Evolut ion Must S ta r t f rom R andom Even ts 467 1.2.2. Ins t ruc t ion Requires In format ion . . . . 467 1.2.3. In format ion Originates or Gains Value by S e l e c t i o n . . . . . . . . . . . . . . . 469 1.2.4. Selection Occurs wi th Special Substances under Special Conditions . . . . . . . . 470

/pdf/selforganization-of-matter-and-the-evolution-of-biological-3ms9rkbxjp.pdf

Selforganization of matter and the evolution of biological macromolecules

Viroids are uncoated infectious RNA molecules pathogenic to certain higher plants. Four different highly purified viroids were studied. By ultracentrifugation, thermal denaturation, electron microscopy, and end group analysis the following features were established: (i) the molecular weight of cucumber pale fruit viroid from tomato is 110,000, of citrus exocortis viroid from Gynura 119,000, of citrus exocortis viroid from tomato 119,000 and of potato spindle tuber viroid from tomato 127,000. (ii) Viroids are single-stranded molecules. (iii) Virods exhibit high thermal stability, cooperativity, and self-complementarity resulting in a rod-like native structure. (iv) Viroids are covalently closed circular RNA molecules.

https://www.pnas.org/content/pnas/73/11/3852.full.pdf

Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures.

RNA folding into stable tertiary structures is remarkably sensitive to the concentrations and types of cations present; an understanding of the physical basis of ion-RNA interactions is therefore a prerequisite for a quantitative accounting of RNA stability. This article summarizes the energetic factors that must be considered when ions interact with two different RNA environments. “Diffuse ions” accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A “chelated” ion directly contacts a specific location on the RNA surface and is held in place by electrostatic forces. Energetic costs of ion chelation include displacement of some of the waters of hydration by the RNA surface and repulsion of diffuse ions. Methods are discussed for computing both the free energy of the set of diffuse ions associated with an RNA and the binding free energies of individual chelated ions. Such calculations quantitatively account for the effects of Mg 2+ on RNA stability where

/pdf/a-guide-to-ions-and-rna-structure-4vheaf1zfz.pdf

A guide to ions and RNA structure

The mechanism of the enhancement of the fluorescence of ethidium bromide on binding to double helical RNA and DNA has been investigated. From an examination of the effect of different solvents on the fluorescence lifetime, quenching of fluorescence by proton acceptors, and the substantial lengthening of lifetime observed upon deuteration of the amino protons, regardless of the medium, we conclude that proton transfer from the excited singlet state is the process primarily responsible for the approximately equal to 3.5-fold increase in the lifetime of free ethidium bromide in going from H2O to D2O; the fact that addition of small amounts of water to nonaqueous solvents decreases the fluorescence whereas addition of small amounts of D2O enhances the fluorescence; and the enhancement of the ethidium bromide triplet state yield on binding to DNA. Other proposed mechanisms are shown to be inconsistent with our findings.

Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids

The problem of how ions influence the folding of RNA into specific tertiary structures is being addressed from both thermodynamic (by how much do different salts affect the free energy change of folding) and structural (how are ions arranged on or near an RNA and what kinds of environments do they occupy) points of view. The challenge is to link these different approaches in a theoretical framework that relates the energetics of ion-RNA interactions to the spatial distribution of ions. This review distinguishes three different kinds of ion environments that differ in the extent of direct ion-RNA contacts and the degree to which the ion hydration is perturbed, and summarizes the current understanding of the way each environment relates to the overall energetics of RNA folding.

Ions and RNA Folding

The binding of Mg2+ to tRNAPhe (yeast) in three conformational states was studied at 10, 30, 45, and 70°C by the fluorescence indicator 8-hydroxyquinoline 5-sulphonic acid in the presence of 0.032 M monovalent cations (Na+). At temperatures below those characteristic for early melting (completely folded tRNA) the Scatchard plots are biphasic. They are well fitted by two classes of noninteracting binding sites with stability constants independent of temperature (KA= 9 × 104, KB= 6 × 103 M−1). In partially unfolded tRNA the strong binding process is co-operative. A single class of weak sites was found in the statistically coiled conformation at 70°C (KB= 3.3 × 103 M−1). The total number of binding sites is 23 ± 5; differences for the folded and unfolded conformations are smaller than 1. The influence of Mg2+ on the stability of the conformational elements of tRNAPhe (yeast) and its CCA-half (i.e. nucleotides 38–76) was determined by differential ultraviolet absorbance and depolarisation melting curves using the fluorescence of the Y base. Tertiary structure corresponding to early melting is stabilized by strongly bound Mg2+, whereas all other melting transitions are only influenced by Mg2+ bound at weak sites. The stability constants of tertiary structure obtained from the melting experiments can quantitatively be described by assuming that 5 ± 1 non-interacting strong sites as characterized by the fluorescence titrations are converted to weak sites upon unfolding of the tertiary structure. Co-operative interaction of Mg2+ with the 5 strong sites in the folded conformation of tRNA can be ruled out. Strong binding of Mg2+ to completely folded tRNA does not produce a conformational transition changing ultraviolet absorbance, circular dichroism and sedimentation coefficient.

tRNA Conformation and Magnesium Binding

A high precision differential absorption technique has been developed for the detailed experimental analysis of the complex melting behaviour of specific tRNAs. This technique allows the resolution of overlapping transitions which have been observed in a study of the interaction of Mg2+ ions with tRNAAla (yeast). On the basis of these experimental observations a strong coupling between two adjacent transitions in tRNAAla is proposed and discussed with respect to the structure of tRNAAla. The sedimentation coefficient of tRNAAla as a function of temperature up to 80° has been studied in detail. The wavelength dependences of the hyperchromicities of various transitions in tRNA and tRNA half molecules are discussed with regard to A · U and G · C pairs melting in these processes. From a comparison of the melting behaviour of tRNAAla, tRNATyr, tRNASer, and tRNAPhe in the presence and the absence of Mg2+ ions it is shown that tRNAAla is best suited for detailed studies of conformational changes in tRNA.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1970.tb00978.x

The coupling of conformational transitions in alanine specific transfer ribonucleic acid from yeast studied by a modified differential absorption technique.

Ethidium bromide destabilizes the low-temperature transitions of tRNAPhe (yeast) and tRNAVal (E. coli), in contrast to its effect upon the double-helix to coil transitions in all other known cases. This fact leads to the interpretation of the low-temperature transition as the unfolding of tertiary structure. Fluorescence temperature-jump experiments show the binding of ethidium bromide to increase as a result of this unfolding. The number of binding sites and the binding constants are n= 3 and K= 2.3 μM−1 in the folded state (8°C) and n= 6 and K= 1.1 μM−1 in the unfolded state (32°C).

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1973.tb02710.x

The binding of ethidium bromide to different conformations of tRNA. Unfolding of tertiary structure.

Kinetics of conformational changes in tRNAPhe (yeast) as studied by the fluorescence of the y-base and of formycin substituted for the 3'-terminal adenine

Conditions were established that allowed the observation of the unfolding of the tertiary structure of tRNAPhe (yeast) without the interference of either secondary structure or low salt aberrant structures. Relaxation kinetics of tertiary structure melting show that the reaction proceeds according to a co-operative all-or-none mechanism. The negative activation enthalpy of formation (ΔH‡R=−14 ± 5 kcal/mol, −59 ± 21 kJ/mol) implies a fast pre-equilibrium preceding the rate-limiting step. The ionic strength dependence of the corresponding rate constant demonstrates that most of the electrostatic repulsion characteristic of tertiary structure folding is overcome before the rate-limiting step is reached. On the other hand, most of the stabilizing enthalpy change occurs after the rate-limiting step. At the usual ionic strength (0.1 M Na±) tertiary structure folding is about 100 times slower than double-helix formation. Extrapolation of the rate constants to high ionic strengths, however, indicates that the dynamic differences between secondary and tertiary structure are only due to electrostatic repulsion. The stabilization of tertiary structure by alkaline salts is increased by decreasing the cationic radius. Double helices show virtually no dependence on the radius of monovalent cations. This indicates considerable geometric restrictions for the stabilization of tertiary structure.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1975.tb02180.x

Roland Römer

Papers

tRNA Conformation and Magnesium Binding

The coupling of conformational transitions in alanine specific transfer ribonucleic acid from yeast studied by a modified differential absorption technique.

The binding of ethidium bromide to different conformations of tRNA. Unfolding of tertiary structure.

Kinetics of conformational changes in tRNAPhe (yeast) as studied by the fluorescence of the y-base and of formycin substituted for the 3'-terminal adenine

Tertiary Structure of tRNAPhe (Yeast): Kinetics and Electrostatic Repulsion