Association of RNA polymerase III with transcription factors in the absence of DNA.

The gene for tRNAMet1 from Xenopus oocytes was transcribed in a cell free system with components isolated from a HeLa cell-free extract. It was found that, apart from the established assembly of transcription factors IIIB and IIIC on tRNA genes into stable transcription complexes, these factors can also associate with the enzyme in the absence of DNA to form a functional polymerase III complex. These complexes can be isolated in a highly active form from the bulk of other cellular proteins by mild methods such as gel filtration or density gradient centrifugation. When associated with RNA polymerase III into a functional complex, the transcription factors IIIB and IIIC can clearly be differentiated from free transcription factors, which individually display a much lower relative molecular mass. The polymerase complexes are stable against 1 M KCl, rendering unlikely that they represent fortuitous aggregates including RNA polymerase III and transcription factors IIIB and IIIC. These complexes are sensitive to dilution and, whereas transcription factor IIIC binds to the enzyme more tightly, factor IIIB tends to leak from the complex upon dilution of the protein concentration. From these results it is clear that in addition to their function as DNA-binding protein(s), transcription factors IIIB and IIIC can directly interact with RNA polymerase III without prior binding to the promoter region of the gene to be transcribed.