Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy

Abstract

The mutual orientations of large and small subunits in the 70 S ribosome have been determined by two types of immune electron microscopic experiments. In a series of single-labeling experiments, sites are mapped on a specific projection of the 70 S ribosome (the non-overlap view) and are compared with their known locations on the large subunit. From this comparison the large subunit was shown to be in the φ = 70 ± 12 ° (θ was indeterminate) orientation. The second type of experiment uses simultaneous labeling of 70 S ribosomes, with antibodies against two ribosomal proteins (one on the large subunit and one on the small subunit) to orient both subunits within the 70 S ribosome. These experiments, designed so that subunits can be oriented without reference to specific projections, showed that proteins L7/L12 and S6 are on opposite sides of the ribosome. In addition, they showed that protein S14 and a determinant, LY (near the nascent chain exit site), are also on opposite sides of the ribosome. These results define the general arrangement of large and small subunits in the ribosome and, given the hand of the small subunit, they fix the hand of the large subunit. The results of both types of experiments support the “external-stalk” 70 S model and both are inconsistent with the two different “internal-stalk” models that have been proposed.