Studies of Ribosomal Diffusion Coefficients Using Laser Light-Scattering Spectroscopy

Abstract

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).