Rrn3 becomes inactivated in the process of ribosomal DNA transcription

The human homologue of yeast Rrn3, a 72-kDa protein, is essential for ribosomal DNA (rDNA) transcription. Although the importance of Rrn3 function in rDNA transcription is well established, its mechanism of action has not been determined. It has been suggested that the phosphorylation of either yeast RNA polymerase I or mammalian Rrn3 regulates the formation of RNA polymerase I.Rrn3 complexes that can interact with the committed template. These and other reported differences would have implications with respect to the mechanism by which Rrn3 functions in transcription. For example, in the yeast rDNA transcription system, Rrn3 might function catalytically, but in the mammalian system it might function stoichiometrically. Thus, we examined the question as to whether Rrn3 functions catalytically or stoichiometrically. We report that mammalian Rrn3 becomes the limiting factor as transcription reactions proceed. Moreover, we demonstrate that Rrn3 is inactivated during the transcription reactions. For example, Rrn3 isolated from a reaction that had undergone transcription cannot activate transcription in a subsequent reaction. We also show that this inactivated Rrn3 not only dissociates from RNA polymerase I, but is not capable of forming a stable complex with RNA polymerase I. Our results indicate that Rrn3 functions stoichiometrically in rDNA transcription and that its ability to associate with RNA polymerase I is lost upon transcription.