Faithful transcription of ribosomal 5-S RNA in vitro depends on the presence of several factors.

Abstract

Cytoplasmic extracts from HeLa cells, capable of transcribing the cloned genes for ribosomal 5-S RNA, were employed to study the factors involved in this process. Two factors can be isolated, by gel filtration through Sephadex G-100, which are devoid of RNA polymerase activity. They significantly enhance the extent and specificity of the transcription of 5-S rRNA. Both proteins can jointly be purified by affinity chromatography on immobilized DNA containing the genes for ribosomal 5-S RNA from Xenopus borealis. Besides a protein of approximately 45 kDa, possibly corresponding to TF IIIA isolated from Xenopus oocytes, a second protein with a molecular mass of 22 +/- 1 kDa stimulates the formation of 5-S RNA. This protein is contained in the breakthrough of DEAE-cellulose; it binds to and is eluted from phosphocellulose with 0.6 M KCl. In addition, it was found that the exclusion volume obtained after gel filtration on Sephadex G-100 contains functional complexes, which are capable of transcribing the cloned 5-S genes and hence contain all the required factors. Direct evidence is presented that the protein of 22 kDa described above is contained in and can be isolated from such complexes. It is postulated from indirect evidence that an additional factor with a molecular mass in excess of 100 kDa is required which can be removed from functional polymerase complexes by gel filtration through Bio-Gel A5m.