Heterogeneity in the 3'-terminal sequence of ribosomal 5S RNA synthesized by isolated HeLa cell nuclei in vitro.

Isolated HeLa cell nuclei synthesize ribosomal 5S RNA of very nearly correct sequence and size. The in vitro product was resolved according to size on formamide-containing polyacrylamide gels and the fractions were subsequently hybridized to recombinant DNA containing the 5S genes from Xenopus mulleri. It could be shown that the 5S RNA synthesized in vitro differed only very slightly in size from the mature species labeled in vivo and contained a few extra nucleotides in some of the molecules. Analysis of the 3'-terminal base of molecules synthesized independently with four different nucleotides showed that the chains were almost exclusively terminated with uridine. Digestion of the in vitro product with ribonuclease T1 and analysis of the oligonucleotides on DEAE-Sephadex A-25 in the presence of 7 M urea revealed that the molar yield of the internal fragments agreed well with the expected theoretical values. The 3'-terminal fragments, however, were found to be present in three different species with the sequences CUUOH, CUUUOH, CUUUUOH which occurred with a frequency of about 60%, 20%, and 20%, respectively. From these data we conclude that 5S RNA synthesis in isolated HeLa cell nuclei was correctly initiated but that termination occurred with a slight ambiguity, adding either one or two uridine residues to some of the chains.