Nascent RNA chain termination and ultrastructural changes in nucleoli isolated from SV40-transformed fibroblasts treated with aminonucleoside of puromycin

The mechanism for the selective inhibition of preribosomal RNA synthesis by puromycin aminonucleoside (AMS) was investigated in cultured AG 2804 cells, which are human embryonic lung fibroblasts transformed by SV40 virus. Sequential high voltage electrophoresis and paper chromatography of the RNA of fractionated nuclei isolated from fibroblasts exposed to tritiated AMS (340 μM, 5 μCi. per ml., for 18 hours) demonstrated that the inhibitor is demethylated to 3'-amino-3'-deoxyadenosine and incorporated into terminal positions in both nucleolar and nonnucleolar nuclear RNA chains in the same relative amounts. 3'-Amino-3'-deoxyadenosine was not detected in the RNA obtained from the cytoplasm of AMS-treated fibroblasts. Electron microscopic observation of isolated nucleoli showed that neighboring intranucleolar fibrils have a tendency to coalesce after AMS treatment. It is suggested that, when the modified AMS molecule has reached the growing ends of nascent polyribonucleotide chains elongation stops, the chain remains attached to its template, and the strong positive charge of the aminosugar portion of AMS interacts with negatively charged components (e.g. phosphate groups) of adjacent nascent RNA chains. The high density of the components of the nucleolus produces frequent interactions of this type and leads to secondary interference with nucleolar RNA synthesis, thus accounting for the selective effect of AMS on preribosomal RNA synthesis.