The HMG box-containing nucleolar transcription factor UBF interacts with a specific subunit of RNA polymerase I.

Abstract

The mammalian transcription activator protein UBF contains five tandemly repeated HMG homology domains which are required for DNA binding. We have used highly purified RNA polymerase I (Pol I) and upstream binding factor (UBF) and investigated whether these two proteins interact in solution. We show by a variety of different experimental approaches, such as immunoprecipitation, glycerol gradient sedimentation, affinity chromatography and protein blotting, that UBF physically associates with Pol I. Mutational analysis reveals that the HMG boxes play an important role in this specific interaction. UBF binds to mouse and yeast Pol I, demonstrating that the interaction of UBF with Pol I has been conserved during evolution. Interestingly, in both species one Pol I‐specific subunit (34.5 kDa in yeast and 62 kDa in mouse) was recognized by UBF. No specific interaction was observed with Pol II. Unexpectedly, UBF was found to associate also with a unique subunit of yeast Pol III. This apparent specific interaction of UBF with the two classes of RNA polymerases may reflect functionally important interactions of HMG box‐containing transcription factors with the transcriptional apparatus.

Cell cycle-dependent regulation of RNA polymerase I transcription: The nucleolar transcription factor UBF is inactive in mitosis and early G 1

“…Although the present data do not definitely exclude any of these possibilities, we favor the latter one for the following reasons. UBF has been shown to interact with a number of proteins, including the retinoblastoma protein pRb (19,28), the third largest subunit of pol I (29,30), and p53 (unpublished data). All of these interactions are mediated by the amino-terminal part of UBF.…”

Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit–subunit contact network involving Rpb5

“…In order to reveal the subunit-subunit contact network involving Rpb5 within the S. pombe RNA polymerase II, we employed far-Western analysis in which S. pombe RNA polymerase II was separated into individual subunits by SDS-PAGE, blotted onto membranes and probed by radio-labelled recombinant Rpb5. Far-Western blot analysis has been successfully employed for detection of molecular interactions between RNA polymerase subunits at least with some transcription factors (Schnapp et al 1994;Cheong et al 1995), indicating that RNA polymerase subunits expose, at least in part, the contact surface with other proteins even after blotting on membranes. In fact, we detected that Rpb5 binds to Rpb1, Rpb2 and Rpb3 (see Figs 2 and 3).…”

Mechanism of Repression of RNA Polymerase I Transcription by the Retinoblastoma Protein

“…The product of the overlap extension PCR was digested with NdeI and BglII and used to replace the first 487 amino acids of pET-His-UBF1 (39). All other plasmids encoding UBF1 and the different UBF mutants have been described before (36,40).…”

“…Glutathione-Sepharose beads bearing either GST alone or the GST-pRb fusions were incubated with either cellular UBF or 35 S-labelled recombinant UBF, which was generated in a coupled in vitro transcriptiontranslation rabbit reticulocyte lysate system (Promega). The UBF mutants used have been described previously (36). Interaction assays were conducted as follows.…”