Mapping of nuclease-sensitive sites in native reticulocyte ribosomes--an analysis of the accessibility of ribosomal RNA to enzymatic cleavage.

Abstract

Treatment of ribosomes in reticulocyte lysates with low concentrations of the calcium-dependent nuclease from Staphylococcus aureus resulted in cleavage of rRNA. The positions of the cleaved phosphodiester bonds were localised by primer extension and polyacrylamide gel electrophoresis. S. aureus nuclease-induced strand scissions were found in the 5'-domain of 18S rRNA and in domains II, IV and VI of 28S rRNA. The majority of the cleavage sites were located in eukaryote-specific expansion segments and only one cleavage site was found in a region suggested to be directly involved in ribosomal function. Treatment of the reticulocyte lysate with increasing amounts of S. aureus nuclease resulted in the introduction of new cleavage sites. However, even at the highest nuclease concentration used, large parts of the rRNAs were protected from nuclease digestion. Removal of translational components, by salt wash of isolated reticulocyte polysomes, exposed additional rRNA sequences to S. aureus nuclease cleavage. These sequences were found in the 3'-major domain of 18S rRNA and in domains II, IV, and V of 28S rRNA. These sites are located at the putative translational surface of the ribosome. The translational activity of the S. aureus nuclease-treated ribosomes, determined after addition of exogenous mRNA, was directly correlated to the extent of nuclease digestion of the ribosomes. However, the decrease in translational activity observed in lysates treated with low amounts of S. aureus nuclease was not due to a preferential exclusion of damaged ribosomes from polysome formation. This suggests that the induced cleavages were not detrimental to ribosomal function but could influence the rate of ribosomal movement along the mRNA.