We demonstrate that the major in vivo targets of brefeldin A (BFA) in the secretory pathway of budding yeast are the three members of the Sec7 domain family of ARF exchange factors: Gea1p and Gea2p (functionally interchangeable) and Sec7p. Specific residues within the Sec7 domain are important for BFA inhibition of ARF exchange activity, since mutations in these residues of Gea1p (sensitive to BFA) and of ARNO (resistant to BFA) reverse the sensitivity of each to BFA in vivo and in vitro. We show that the target of BFA inhibition of ARF exchange activity is an ARF-GDP-Sec7 domain protein complex, and that BFA acts to stabilize this complex to a greater extent for a BFA-sensitive Sec7 domain than for a resistant one.

While initiation of transcription by RNA polymerase III (Pol III) has been thoroughly investigated, molecular mechanisms driving transcription termination remain poorly understood. Here we describe how the characterization of the in vitro transcriptional properties of a Pol III variant (Pol IIIdelta), lacking the C11, C37, and C53 subunits, revealed crucial information about the mechanisms of Pol III termination and reinitiation. The specific requirement for the C37-C53 complex in terminator recognition was determined. This complex was demonstrated to slow down elongation by the enzyme, adding to the evidence implicating the elongation rate as a critical determinant of correct terminator recognition. In addition, the presence of the C37-C53 complex required the simultaneous addition of C11 to Pol IIIdelta for the enzyme to reinitiate after the first round of transcription, thus uncovering a role for polymerase subunits in the facilitated recycling process. Interestingly, we demonstrated that the role of C11 in recycling was independent of its role in RNA cleavage. The data presented allowed us to propose a model of Pol III termination and its links to reinitiation.

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, hTAF II 68. 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 (TAF II s). In vitro binding studies revealed that both EWS and hTAF II 68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAF II 68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAF II 68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAF II 68 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.Structural alteration or aberrant expression of transcription factors is often a critical event in tumorigenic transformation (13,19, 22). Karyotypic analysis has revealed a tumor-specific t(11;22)(q24;q12) chromosomal translocation in 86% of both Ewing sarcoma and primitive neuroectodermal tumor, suggesting that the product of this rearrangement is involved in the formation of these malignancies (34). This chromosomal translocation fuses the EWS gene on chromosome 22 to the FLI-1 gene on chromosome 11 (8). EWS is a protein with unknown function containing an RNA-binding motif and an activation domain(s) (18,24,25). In the EWS-FLI-1 fusion protein, the RNA-binding motif containing the C-terminal half of EWS is replaced by the DNA-binding domain (DBD) of the FLI-1 protein. FLI-1 is a member of the ETS family of transcription factors which activate specific target genes by binding to their cognate DNA sequences through their DNA-binding regions, usually located at their carboxyl termini (2, 37). The replacement of the native transcription activation domain(s) of FLI-1 by the N-terminal region of EWS converts the nontransforming activator, FLI-1, into a transforming protein with new transcriptional activation potential. In the EWS-FLI-1 fusion protein, both the N-terminal domain of EWS and the DBD of FLI-1 are necessary for the transforming activity (20). Recently, the EWS gene was also shown to be involved in tumorigenesis by chromosomal translocation with other genes encoding either other members of the ETS family (Erg, ETV1, E1A-F, and FEV) or other transcription factors, including ATF-1, WT1, and the nuclear orphan receptor TEC1 (...