Heterogeneity of Escherichia coli ribosomes established by scanning transmission electron microscopy

Abstract

Quantitative mass image analysis of Escherichia coli ribosomal particles by scanning transmission electron microscopy (STEM) provided direct evidence that presumably homogeneous preparations of ribosomes are, in reality, populations of heterogeneous particles. Variations in composition, relative molecular mass ( M r) and shape were observed both in the monosomes and in the ribosomal subunits. None of these changes can be resolved visually; they can be evaluated only by computer processing. The variations in relative mass and shape monitored by values of radius of gyration ( R G) were attributed to the loss of ribosomal proteins and/or factors and correlated with the changes in ribosome composition and biological activity. The highest activity was found in monosomes prepared from the standard 0.5 M NH 4Cl wash. With increasing concentrations (up to 1.5 M) of NH 4Cl in the wash buffer the activity decreased slowly, then dropped rapidly to about half in 2 M NH 4Cl. The most striking effects were observed in ribosomal particles washed with 0.1 M NH 4Cl. The 70S monosomes and the 30S subunits attained maximum M r and R G values (2660 kDa and 76 Å, and 990 kDa and 75 Å, respectively), which were greater than the theoretical values, while the activity was minimal (∼ 12%). The M r and R G parameters of the 50S subunits remained uneffected by the NH 4Cl washes (∼ 1600 kDa and 68 Å).