Transcription complexes for various class III genes differ in parameters of formation and stability towards salt

RNA polymerase III faithfully transcribes the genes for ribosomal 5 S RNA, tRNA(1Met) or adenovirus VA RNA in vitro in the presence of required transcription factors. These genes display distinct differences in the kinetics of transcription complex formation and in their response to excess template. In contrast to tRNA and VA RNA synthesis, 5 S RNA synthesis displays a lag phase of 15 minutes before the onset of transcription and is clearly inhibited by high concentrations of template. Once formed, transcription complexes for the RNA polymerase III genes listed can be isolated by glycerol gradient centrifugation and display a remarkable stability against transient treatment with high salt concentrations. Complexes for 5 S RNA and tRNA remain functionally active up to 2.5 M-KCl. The activity of transcription complexes for VA RNA, however, is significantly diminished after treatment with high salt concentrations. This effect is shown to be due to an irreversible loss of transcription factors. RNA polymerase III is dissociated by high salt concentrations from all the transcription complexes studied but remains part of these complexes during the normal reinitiation cycle at 60 mM-KCl. An additional method for the purification of partial transcription complexes was developed that involves equilibrium centrifugation on cesium sulfate gradients. This method completely releases TFIIIB from 5 S complexes and a core complex, composed of the 5 S RNA gene, factors IIIA and IIIC, is retained. In the case of tRNA and VA RNA, core complexes are obtained that remain partly associated with TFIIIC and TFIIIB. These results indicate a qualitatively and/or quantitatively different interaction of individual factors in different polymerase III transcription complexes.