Identification of a novel 70 kDa protein that binds to the core promoter element and is essential for ribosomal DNA transcription

Abstract

Mammalian ribosomal RNA genes (rDNA) are transcribed by RNA polymerase I and at least two auxiliary factors, UBF and SL1/TFID/TIF-IB. It has also been reported that an additional factor(s) is required to reconstitute efficient initiation of rDNA transcription in vitro, depending upon the procedures of chromatographic separation. In an attempt to elucidate the molecular identity of such yet uncertain activities, we have developed agarose gel shift and UV cross-linking assays to detect proteins directly bound to the core promoter region of murine rDNA. With these techniques, we identified a 70 kDa protein (p70) in the flow-through fraction of a phosphocellulose column (TFIA-fraction). Interestingly, the binding of p70 to the rDNA core promoter was observed only in the presence of the SL1-containing fraction. The probable human orthologue of p70 was also detected in HeLa cells. Consistent with the observation that p70 bound to the core promoter only in the presence of the TFIA- and SL1-fractions, alteration of DNase I footprint pattern over the core promoter element was demonstrated by cooperative action of the TFIA- and SL1-fractions. A reconstituted in vitro transcription assay with further purified p70 indicated that p70 was required for accurate initiation of rDNA transcription. These results indicate that the p70 identified recently by the current DNA-binding experiments represents a novel transcription factor in rDNA transcription.