Showing papers by "Laszlo Tora published in 1998"
TL;DR: It is demonstrated that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs) and subunits of Pol II have been identified, confirming the association with the polymerase.
Abstract: The t(11;22) chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminus-encoding region of the EWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI-1 gene. As the function of the protein encoded by the EWS gene remains unknown, we investigated the putative role of EWS in RNA polymerase II (Pol II) transcription by comparing its activity with that of its structural homolog, hTAFII68. We demonstrate that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs). In vitro binding studies revealed that both EWS and hTAFII68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAFII68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAFII68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAFII68 have been identified, confirming the association with the polymerase. In contrast to EWS, the tumorigenic EWS–FLI-1 fusion protein is not associated with either TFIID or Pol II in Ewing cell nuclear extracts. These observations suggest that EWS and EWS–FLI-1 may play different roles in Pol II transcription.
262 citations
TL;DR: The results indicate that TBP-free RNA polymerase II mediated transcription may be able to occur in mammalian cells and that multiple preinitiation complexes may play an important role in regulating gene expression.
Abstract: Initiation of transcription of a gene from a core promoter region by RNA polymerase II requires the assembly of several initiation factors to form a preinitiation complex. Assembly of this complex1,2 is thought to be nucleated exclusively by the sequence-specific binding of the TFIID transcription factor complex, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs) (refs 3, 4), to the different promoters. Here we isolate and characterize a new multiprotein complex that does not contain either TBP or a TBP-like factor but is composed of several TAFIIs and other proteins. This complex can replace TFIID on both TATA-containing and TATA-lacking promoters in in vitro transcription assays. Moreover, an anti-TBP antibody that inhibits TBP- and TFIID-dependent transcription does not inhibit activity of this new complex. These results indicate that TBP-free RNA polymerase II mediated transcription may be able to occur in mammalian cells and that multiple preinitiation complexes may play an important role in regulating gene expression.
243 citations
TL;DR: It is reported here that TIF1α is both a phosphoprotein and a protein kinase that undergoes ligand-dependent hyperphosphorylation as a consequence of nuclear receptor binding and that purified recombinant Tif1α possesses intrinsic kinase activity.
71 citations
TL;DR: The results suggest that ER acts, in an early step, during or immediately after the formation of template-committed complexes containing TFIIB, favouring the recruitment of one or more components of the basic transcription machinery as well as co-activators.
Abstract: The action of oestrogen hormones is mediated through the oestrogen receptor (ER), a member of a large superfamily of nuclear receptors that function as ligand-activated transcription factors. Sequence-specific transcription factors, including the nuclear receptor superfamily, are thought to interact either directly or indirectly with general transcription factors to regulate transcription. Although numerous studies have focused on the identification of potential co-activators interacting with isolated trans-activation domains of ER, few have investigated the mechanisms by which ER transmits its signal to the basal transcription machinery. We show that ER does not stabilize the binding of the TATA-box binding protein (TBP) of the TFIID complex, or of TFIIB to the promoter, although a stable ER-TBP-TFIIB-promoter complex was detected, suggesting that ER, TBP and TFIIB might interact with each other to form a complex to the promoter. We also demonstrate that ER binds specifically to TFIIB, a key component of the preinitiation complex. Affinity chromatography with immobilized deletion mutants of ER maps a TFIIB interaction region that encompasses the DNA-binding domain. The addition of excess TFIIB to transcription reactions in vitro did not, however, affect the magnitude of transcriptional activation by ER. These results indicate that, in contrast with current models, ER does not activate transcription by increasing the rate of assembly of TFIIB into the transcription complex. An increased concentration of TFIIB was unable, by itself, to overcome the requirement for ER. By using an immobilized promoter-template assay employing nuclear extract from HeLa cells, recombinant human ER increased the stable association of subsequent components of the transcription machinery (TFIIE and TFIIF), in correlation with ER-induced transcription. Our results suggest that ER acts, in an early step, during or immediately after the formation of template-committed complexes containing TFIIB, favouring the recruitment of one or more components of the basic transcription machinery as well as co-activators.
35 citations