Effects of magnesium and ionic strength on the diffusion and charge properties of several single tRNA species.

The technique of laser light scattering was used to evaluate the effects of Mg+2 and ionic strength on the solution structures of seven tRNA species. Information about ion effects on both conformation and electric charge were derived from measurements of the translational diffusion constants and diffusive virial coefficients. E. coli tRNAMetf and six elongator tRNAs from both Class I and II were studied. The diffusion measurements show that the responses of all but the initiator species are qualitatively similar to each other and to that of bulk tRNA, but that significant quantitative differences also obtain. All of the elongator species exhibited an anomolous increase in diffusivity reported earlier by us for bulk tRNA when placed in a low salt-low Mg+2 condition. The initiator tRNA did not undergo this transition and unlike the other tRNAs tested was apparently more compact in 1 mM Mg+2 than 10 mM Mg+2 at ionic strengths in excess of 0.1 M. At 0.1 M ionic strength, pH 7.2, the average net charge of the tRNAs ranged from 7-12 e- in 1 mM Mg+2 and 3-7 e- in 10 mM Mg+2, consistent with the binding of 1-2 additional Mg+2 ions in the higher Mg+2 condition.

Changes in the solution structure of yeast phenylalanine transfer ribonucleic acid associated with aminoacylation and magnesium binding.

The effect of aminoacylation on the structure of yeast phenylalanine tRNA was evaluated by laser light scattering. In these experiments, the translational diffusion coefficient (D20,w) of phenylalanyl-tRNA was monitored continuously during spontaneous deacylation in a variety of solution conditions. The results reveal that significant changes can occur in the hydrodynamic volume and electric charge as a consequence of aminoacylation but that the effects are magnesium dependent. At neutral pH, 20 degrees C, and 0.1 M salt, the D20,w value increased by 18% when deacylation occurred in 2--10 mM Mg2+ concentrations while no change in diffusivity was observed for tRNA deacylating in 0.5--1.0 mM Mg2+. The Mg2+ concentration dependence of the D20,w changes behaves in highly cooperative manner. The electric charges of aminoacyl-tRNA and nonacylated tRNA in 1 and 10 mM Mg2+ were estimated from the diffusive virial coefficients. In the higher Mg2+ conditions, aminoacyl-tRNA has a charge of 15 +/- 2e- while that of the nonacylated form is 10 +/- 2e-; both acylated and nonacylated tRNA have a charge of 11 +/- 4e- in 1 mM Mg2+. Taken together, the results indicate that aminoacylation permits the binding of additional Mg2+, resulting, in turn, in the formation of a more extended conformer of lower diffusivity and greater negative charge. The results also provide a possible explanation for several contradictory results in the literature.

Detection of ligand-induced conformational changes in phenylalanyl-tRNA synthetase of Escherichia coli K10 by laser light scattering.

The diffusion constant of phenylalanyl-tRNA synthetase has been measured by laser light scattering under conditions of complex formation with Mg2+, L-phenylalanine, MgATP, tRNAPhe, modified tRNAPhe, tRNAPhe (yeast), and noncognate tRNA. The diffusion constant (pH 7.5, 20 degrees C) of the free enzyme is (2.85 +/- 0.005) x 10(-7) cm2 s-1, of the enzyme . Mg2+ complex (2.40 +/- 0.05) x 10(-7) cm2 s-1 and of the enzyme . Mg2+ . tRNAPhe complex (2.95 +/- 0.06) x 10(-7) cm2 s-1. The effect of tRNAPhe is only seen when the enzyme is saturated with Mg2+. The smaller substrates exhibit no effect besides a small increase of the value of the diffusion constant under conditions where the enzyme-phenylalanyladenylate is synthesized. Of the noncognate tRNATyr and tRNAIle, the latter is able to associate with the enzyme, causing the value of the diffusion constant to increase. tRNAPhe (yeast) and tRNAhvPhe (photo-cross-linked tRNAPhe) exhibit similar effects. The observed variation of the diffusion constant is attributed to conformational changes of the enzyme. The opposite effects of Mg2+ and tRNAPhe are interpreted as an expansion and recontraction, respectively, of the enzyme molecule. In several cases, the effects were used to follow a titration of the enzyme with a ligand. Dissociation constants were calculated from the resulting titration curves, yielding values which are in agreement with those obtained by other techniques. It is established by comparison that of the two possible binding sites for each Mg2+ and tRNAPhe the diffusion constant reflects occupation of only a single class of sites.

Conformational changes in 16S ribosomal RNA induced by 30S ribosomal subunit proteins from Escherichia coli.

Laser light scattering has been used to evaluate conformational differences between free 16S RNA and several specific protein-16S RNA complexes. Proteins that interact strongly with the 16S RNA early in subunit assembly stabilize the RNA chain against unfolding in 1 mM Mg2+ and actually promote the formation of a more compact teriary structure in 20 mM Mg2+. A vital function of these proteins may therfore consist in altering the configuration of the RNA so that further assembly reactions can take place.

Laser light-scattering analysis of protoplasmic streaming in the slime mold Physarum polycephalum.

Laser light scattering is shown to be an effective means of obtaining a rapid, objective assessment of dynamic changes in the intact plasmodium of the myxomycete Physarum polycephalum during bidirectional (shuttle) streaming. The motion of material in a 100 mum diameter region of a plasmodial vein was studied by following changes in the autocorrelation function of the fluctuations in the scattered light intensity. The autocorrelation function was recorded at 10 s intervals and analyzed to follow changes in the flow velocity of protoplasm associated with shuttle streaming. Rhythmic velocity changes and a "beating" pattern of velocity maxima were readily observed. In an attempt to locate the site of underlying structural changes in the vein responsible for the changing pattern of flow, the average scattered intensity was separated into components derived from moving and stationary scatterers. Periodic variations in the light intensity due to stationary scatterers are related to the streaming cycle and indicate the occurrence of important structural changes in the vein walls. Two possible interpretations of the data are offered; one involving gross dynamic changes in vein structure, the other involving the formation, contraction, or breakdown of fibrillar material in the vein wall during the streaming cycle.

Studies of ribosomal diffusion coefficients using laser light-scattering spectroscopy.

Using an optical beating technique, the diffusion coefficients and relative scattered intensity of Escherichia coli 70S, 50S, and 30S ribosomes are measured as a function of temperature and Mg(2+) concentration. For solutions at 10 mM Mg(2+) and between 0 degrees C and about 40 degrees C, the values of D(20,w) obtained are 1.7, 1.9, and approximately 2.1 x 10(-7) cm(2)/s, respectively. Preparative procedures drastically affect these values and equivalent hydrodynamic ellipsoids of revolution models give large axial ratios indicating extensive hydration or a deviation from the assumed shape. Calculations also indicate that the subunits expand upon dissociation. Measurements of D(20,w) vs. temperature indicate that 70S particles undergo a conformational change prior to dissociation and can be heat dissociated at 30-32 degrees C at low concentrations. Treatment of 70S ribosomes with EDTA causes a biphasic dissociation reaction. Addition of Mg(2+) after dissociation with EDTA shows that longer waiting times yield fewer 70S particles and that even short waiting times may yield ribosomes differing from the native conformation. Addition of p-chloromercuribenzoic acid (PCMB) is shown to dissociate 70S particles, but to a lesser extent than ethylenediaminetetraacetic acid (EDTA).