A polymerase activity forming 5S and pre-4S RNA in isolated HeLa cell nuclei

Abstract

Isolated HeLa cell nuclei are capable of synthesizing 5S and pre-4S RNA. The labeling of these low molecular weight species has been compared with the labeling of nucleolar RNA and nuclear heterogeneous RNA. The 5S and pre-4S RNA molecules made in vitro were identified by their mobility on SDS acrylamide gels and by the sensitivity of pre-4S RNA to enzymes which cleave it in vitro to 4S RNA. Their mobilities and cleavage properties are similar to the RNA made in vivo. Unlike the nuclear heterogeneous RNA, the synthesis of the two small molecular weight RNAs is resistant to α-amanitin. A large proportion of 4S RNA labeled in vitro appears to be formed de novo. The ratio of the terminal uridine to the internal uridine 3′-monophosphate remains constant with time, even though there is linear incorporation into the pre-4S RNA in the isolated nuclei. Production of the nucleolar RNA and pre-4S RNA has been compared in the presence of various ions. The pre-4S RNA synthesis has a sharper maximum for (NH 4)SO 4 and MgCl 2 than does the synthesis of nucleolar RNA. The in vitro synthesis of pre-4S is more sensitive to ellipticine and pCMB than the production of nucleolar RNA. These differences between the production of pre-4S RNA and nucleolar RNA are discussed with respect to in vitro reinitiation and the possibility that different polymerases are involved in their synthesis.