E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription.

Abstract

We previously described the purification and characterization of EBF, a rat rRNA gene core promoterbinding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of EjBF was selectively phosphorylated. The labeled phosphate could be removed from the EBF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase.Elution of the protein from the ElBF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with [r-32P] Ribosomal RNA represents as much as 80% of total RNA in higher eukaryotes and its synthesis is highly regulated in response to a variety ofphysiological and pathological stimuli (for reviews, see refs. 1 and 2). Transcription of rRNA genes (rDNA) is directed by RNA polymerase I and other accessory transcription factors. E1BF is a factor that can interact with an upstream enhancer element (El) (3,4) as well as the promoter sequence and can stimulate rat rDNA transcription in vitro (5). Recent studies have demonstrated that it can also activate the human rDNA promoter, but not polymerase II promoters (H.N. and S.T.J., unpublished data). When analyzed by SDS/PAGE under reducing conditions, purified E1BF displays two silver-stained bands of Mr 89,000 and 79,000. However, the mechanism by which E1BF modulates rDNA transcription has not been elucidated.Phosphorylation of certain protein factors can modify RNA polymerase II-directed transcription (6-11). Similarly, the functions of growth factor receptors (12) and oncogene products (13) (14). In contrast to polymerase II transcription, phosphorylation of a specific factor in the polymerase I-directed transcription of rDNA has not been reported. Several years ago, our laboratory showed that RNA polymerase I can be activated by purified protein kinase NII (15), which can stimulate polymerase I activity in a filter binding assay (16). To date, however, neither we nor others have been able to demonstrate that phosphorylation of RNA polymerase I is essential for rDNA transcription. Preliminary studies suggested to us that E1BF might be posttranslationally modified. We postulated that this modification might be caused by phosphorylation of the factor. To determine whether E1BF is regulated by phosphorylation/dephosphorylation and to identify the protein kinase that might regulate polymerase I-directed transcription, we initiated a series of experiments. These studies demonstrated that (i) E1BF can indeed be phosphorylated by a protein kinase intrinsic to this factor, (ii) this phosphorylation is confined to the 79-kDa polypeptide of E1BF, and (iii) the phosphatase-induced inactivation of polymerase I transcription can be restored by the addition of purified E1BF. METHODSPurification of E1BF. E1BF was purified from rat mammary adenocarcinoma cell extract as described (5). Purification by the oligonucleotide...