Efficient transcription by RNA polymerase I using recombinant core factor

Transcription of ribosomal DNA by RNA polymerase I is a central feature of eukaryotic ribosome biogenesis. Since ribosome synthesis is closely linked to cell proliferation, there is a need to define the molecular mechanisms that control transcription by RNA polymerase I. To fully define the factors that control RNA polymerase I activity, biochemical analyses using purified transcription factors are essential. Although such assays exist, one limitation is the low abundance and difficult purification strategies required for some of the essential transcription factors for RNA polymerase I. Here, we describe a new method for expression and purification of the three subunit core factor complex from Escherichia coli. We demonstrate that the recombinant material is more active than yeast-derived core factor in assays for RNA polymerase I transcription in vitro. Finally, we use recombinant core factor to differentiate between two opposing models for the role of the TATA-binding protein in transcription by RNA polymerase I.

► Co-expression of Rrn6p, Rrn7p and Rrn11p in E. coli produces an active complex.
► Recombinant core factor is more active that core factor purified from yeast.
► Eukaryotic-specific covalent modifications are not required for core factor activity.
► Tata-binding protein can activate Pol I transcription, without UAF, in vitro.