Efficient transcription by RNA polymerase I using recombinant core factor

Abstract

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.

Figure 1

Core factor assembles in E. coli and is purified as a complex. (A) Purification strategy used to obtain pure rCF. (B) Fractions from monoQ gradient were electrophoresed on an 8% SDS-PAGE gel and visualized with Coomasie Blue stain. Fraction numbers are indicated, and the protein molecular weight marker is labeled on the left.

Figure 2

Recombinant core factor is more concentrated than that purified from yeast. (A) Three dilutions of rCF and yCF were run on a 10% SDS PAGE gel and stained with Sypro Ruby Red. The gel was then scanned using a Typhoon fluorescent scanner (excitation, 488 nm; 610 nm emission filter). Two versions of the same scanned gel are shown. Lower image is a higher contrast version with contrast enhancement performed using ImageQuant software (GE, Uppsala, Sweden). Degradation products or impurities are indicated by asterisks. Rrn11 is slightly larger due to an N-terminal his₆ tag in rCF, and Rrn7 is larger in yCF due to an N-terminal triple hemagluttinin (HA) tag. In both preparations, Rrn11 and Rrn7 ran as doublets. Every preparation of core factor contains visible Rrn6 degradation products. (B) Rrn6 protein abundance was measured using ImageQuant software. Resulting values were multiplied by their dilution factor, averaged and plotted. Error indicated is one standard deviation + and −.

Figure 3

Recombinant core factor has a higher specific activity than yeast-derived core factor. (A) Serial dilutions of yCF and rCF were added to transcription reactions that contained template DNA, UAF, TBP, Pol I and Rrn3. After addition of core factor to the reactions, transcription was initiated by addition of nucleoside triphosphates, including α³²P-GTP. Multiple rounds of transcription were permitted and reactions were halted after 15 min by addition of excess phenol. Transcripts were analyzed on denaturing polyacrylamide gels and quantified using a Storm 820 phosphorimager and ImageQuant software (GE, Uppsala, Sweden). Addition of heat-inactivated rCF to the reactions (H.I.) resulted in no transcript accumulation. (B) Product accumulation was measured for all lanes. The resulting values were multiplied by their dilution factor and averaged. Standard deviation of averaged values was calculated. Relative core factor concentration (from Figure 2) and relative transcription per volume of core factor preparation were calculated and are shown. Propagation of error of these values is indicated. Specific activity was calculated as a ratio of these values, again with propagated error indicated.

Figure 4

Core factor is required for transcription in vitro; TBP and UAF enhance activity. Indicated dilutions of rCF were added to in vitro transcription assays with and without UAF and TBP. The gel running conditions were the same as for Figure 3. The −UAF − TBP sample exhibited basal levels of Pol I transcription, whereas the presence of both UAF and TBP induced maximal promoter-specific transcription. The −UAF +TBP sample yielded more product than − UAF −TBP, demonstrating that TBP can activate Pol I transcription without UAF. Lane numbers are indicated for ease of discussion.