Transcription of the Cloned Genes for Ribosomal 5‐S RNA in a System Reconstituted in vitro from HeLa Cells

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Abstract
Cytoplasmic extracts from HeLa cells, containing transcription factors and intact RNA polymerase C, were used to transcribe the cloned genes for ribosomal 5‐S RNA from Xenopus borealis. It was found that the endogenous enzyme in these extracts synthesizes discrete products of 5‐S size which hybridize predominantly to the 5‐S RNA gene region and reveal an oligonucleotide pattern characteristic of authentic 5‐S ribosomal RNA. The overall transcription of the plasmic DNA is greatly repressed in the presence of the cytoplasmic extract. The specific production of 5‐S RNA by the cytoplasmic extract depends on an optimal DNA concentration, beyond which the excess DNA is transcribed unspecifically. The crude extracts contain a factor which can be enriched by chromatography on phosphocellulose and which binds to DNA. In the presence of the cytoplasmic extract, this factor significantly stimulates 5‐S RNA synthesis at high DNA concentration. No stimulation is observed at limiting DNA concentrations, already saturated by endogenously contained factor. The synthesis of 5‐S RNA in this system depends on the appropriate template since it is not observed with the cloned genes for 5‐S from Drosophila.The cytoplasmic extracts endogenously contain a limited capacity to transcribe the genes for ribosomal 5‐S RNA. This process can be stimulated significantly by the addition of optimal concentrations of purified RNA polymerase C, which by itself transcribes these genes in an entirely random fashion. Reconstitution of plasmid DNA with the DNA‐binding protein and purified polymerase does not lead to the production of 5‐S RNA. From these data we conclude that the cytoplasmic extracts contain additional factor(s) which are deficient in the purified polymerase molecule and which are required together with the DNA‐binding protein for the specific synthesis of ribosomal 5‐S RNA.The observed stimulation of 5‐S RNA synthesis is not seen with RNA poiymerase C from yeast cells, indicating an evolutionary divergence of functional significance in the polymerase molecule.