Isolation of a transcription complex for ribosomal 5S RNA

Cloned 5S rRNA genes from Xenopus borealis oocytes can be used to assemble functional transcription complexes from cytoplasmic HeLa cell extracts as a source for polymerase III and all factors additionally required for faithful 5S RNA transcription. Such complexes can be isolated by glycerol gradient ultracentrifugation and non-denaturing gel electrophoresis. They contain less than 1% of the cellular protein and retain their fidelity to synthesize 5S rRNA. The assembly of the complex is unaffected by KCl concentrations up to 140 mM whereas the transcription of 5S rRNA by the isolated complex is significantly reduced at this ionic strength. This indicates that the latter process, involving re-initiation by RNA polymerase III, is more sensitive to elevated salt concentrations than is the assembly of the transcription complexes. Furthermore, we show that complex formation also takes place in the absence of exogenously added nucleoside triphosphates, although this results in a slight shift in the sedimentation position which can be reversed by addition of the initial nucleotides GTP and CTP. We have analyzed the isolated transcription complexes by the protein blotting technique in an attempt to characterize their DNA-binding components. The results show a single component, corresponding to a protein with a mol. wt. of approximately 45 kd, which binds selectively, but not exclusively to a DNA fragment containing the 5S gene. The possible relationship of this protein to transcription factor IIIA from Xenopus oocytes is discussed.