Mammalian Rrn3 is required for the formation of a transcription competent preinitiation complex containing RNA polymerase I

Mammalian Rrn3, an essential, polymerase-associated protein, is inactivated when cells are treated with cycloheximide, resulting in the inhibition of transcription by RNA polymerase I. Although Rrn3 is essential for transcription, its function in rDNA transcription has not been determined. For example, it is unclear whether Rrn3 is required for initiation or elongation by RNA polymerase I. Rrn3 has been shown to interact with the 43-kDa subunit of RNA polymerase I and with two of the subunits of SL1. In the current model for transcription, Rrn3 functions to recruit RNA polymerase I to the committed complex formed by SL1 and the rDNA promoter. To examine the question as to whether Rrn3 is required for the recruitment of RNA polymerase I to the template, we developed a novel assay similar to chromatin immunoprecipitation assays. We found that RNA polymerase I can be recruited to a template in the absence of active Rrn3. However, that complex will not initiate transcription, even after Rrn3 is added to the reaction. Interestingly, the complex that forms in the presence of active Rrn3 is biochemically distinguishable from that which forms in the absence of active Rrn3. For example, the functional complex is fivefold more resistant to heparin than that which forms in the absence of Rrn3. Our data demonstrate that Rrn3 must be present when the committed template complex is forming for transcription to occur.