The effect of pancreatic ribonuclease on rabbit reticulocyte ribosomes and its interpretation in terms of ribosome structure

Abstract

1. Parts of the 16s and 30s RNA species of reticulocytes are readily hydrolysed by pancreatic ribonuclease. The biological activity of the ribosomes is diminished after treatment with low concentrations of the enzyme (e.g. 1ng. of ribonuclease/2·5mg. of polyribosome fraction/ml.). A high proportion of the chain scissions are ‘hidden’ owing to the secondary structure of the RNA moiety. 2. As the concentration of ribonuclease is increased RNA is lost from the ribosome. About 20–30% of the RNA may be removed from the ribosome without altering appreciably its sedimentation coefficient or its appearance in the electron microscope. 3. The amount of RNA removed from the ribosome is not increased by raising the concentration of enzyme from about 1μg. to 2·5mg. of ribonuclease/2·5mg. of polyribosome fraction/ml., or by increasing the temperature from 0° to 30°, or by first converting the RNA moiety into a single-stranded form before exposure to ribonuclease. 4. Untreated polyribosomes aggregate at about 75°, whereas ribosomes treated with ribonuclease aggregate at about 45°. The aggregates that are found on heating ribosomes after enzymic hydrolysis contain about 40–50% of the complement of RNA of intact ribosomes. 5. From the size of the fragments of RNA isolated from RNA-depleted ribosomes it is inferred that there is one site/60–100 nucleotides that is sensitive to ribonuclease. 6. The RNA moiety of RNA-depleted ribosomes has some double-helical character as shown by the optical properties and X-ray-diffraction pattern of ribonuclease-treated ribosomes and by the ‘melting’ properties of the isolated RNA. 7. Subparticles prepared by titration with an excess of EDTA are readily hydrolysed by ribonuclease to fragments of S_20,w less than 4s, in contrast with the intact particle.