The general transcription factor IIB (TFIIB) is required for transcription of class II genes by RNA polymerase II. Previous studies demonstrated that mutations in the Saccharomyces cerevisiae SUA7 gene, which encodes TFIIB, can alter transcription initiation patterns in vivo. To further delineate the functional domain and residues of TFIIB involved in transcription start site utilization, a genetic selection was used to isolate S. cerevisiae TFIIB mutants exhibiting downstream shifts in transcription initiation in vivo. Both dominant and recessive mutations conferring downstream shifts were identified at multiple positions within a highly conserved homology block in the N-terminal region of the protein. The TFIIB mutations conferred downstream shifts in transcription initiation at the ADH1 and CYC1 promoters, whereas no significant shifts were observed at the HIS3 promoter. Analysis of a series of ADH1-HIS3 hybrid promoters and variant ADH1 and HIS3 promoters containing insertions, deletions, or site-directed base substitutions revealed that the feature that renders a promoter sensitive to TFIIB mutations is the sequence in the immediate vicinity of the normal start sites. We discuss these results in light of possible models for the mechanism of start site utilization by S. cerevisiae RNA polymerase II and the role played by TFIIB.Accurate and efficient transcription of eukaryotic proteincoding (class II) genes involves the concerted action of RNA polymerase II (RNAPII) and a host of accessory proteins. A subset of these proteins are known as the general transcription factors (GTFs) and include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in reference 34). The GTFs are being intensively studied with the objective of determining their respective functions during the different stages of RNAPII transcription, which include (i) formation of a preinitiation complex (PIC) on the promoter, (ii) melting of the promoter DNA, (iii) transcription initiation, (iv) clearance of RNAPII from the promoter, (v) elongation of the nascent transcript, and (vi) transcription termination.Most promoters of class II genes contain both upstream regulatory elements and TATA elements. TATA elements, containing the consensus sequence TATAa/tAa/t, are located upstream of the mRNA start sites and are specific binding sites for the TATA-binding protein (TBP) subunit of TFIID (32, 36). For most class II promoters, formation of an active PIC is thought to occur by the initial binding of TFIID to the TATA element, in some cases accompanied by TFIIA. It is proposed that PIC formation then proceeds by either an ordered stepwise association of the remaining factors and RNAPII or by the direct recruitment of RNAPII holoenzyme (reviewed in reference 34). Upon PIC formation, the promoter DNA can be melted in an energy-dependent step, facilitating the initiation of mRNA synthesis and clearance of RNAPII from the promoter. In higher eukaryotes, transcription initiation usually occurs at a discrete start site located about 25 to 30 bp downstrea...

The human EVI5 gene was originally isolated through its involvement with a constitutional chromosome translocation in a patient with stage 4S neuroblastoma. Recently, it has been shown that EVI5 is a centrosomal protein in interphase cells, which relocalizes to the midbody during late phases of mitosis. Disruption of its function leads to incomplete cell division and the formation of multinucleate cells. The EVI5 protein contains a TBC (TRE2/BUB/ CDC16 homology) motif located in the N-terminal region. Proteins containing a TBC domain have been shown in some cases to act as GTPase-activating proteins (GAPs) and function through the interaction with Rab-like small G proteins. Despite the identification of over 50 TBCcontaining proteins, and over 70 Rab-like proteins, only three combinations have been shown to have Rab/GAP activity to date. In this study, using linear ion trap mass spectroscopy, we have demonstrated that EVI5 exists in a protein complex with Rab11. Further, using a specific Rab-binding assay, we have shown that EVI5 preferentially interacts with the guanosine triphosphate-bound form of Rab11, and in a GAP activity assay, we have confirmed that EVI5 functions as a GAP for the Rab11 GTPase.Oncogene ( Proteins with homology to the so-called TBC domain, consisting of an B200-amino-acid motif initially identified in the TRE2/BUB2/CDC16 genes (Richardson and Zon, 1995), are considered to function as GTPaseactivating proteins (GAPs), partnering with Rab-like small G proteins. Rab GTPases are members of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins that play critical roles in the regulation of important membrane and protein trafficking events in the cell. Many of the Rab proteins are associated with fundamental biological processes such as vesicle fusion, receptor recycling, membrane transport and cytokinesis (Zerial and McBride, 2001). There are, however, over 50 human proteins with predicted TBC domains, and over 70 human Rab proteins, of which only two TBC domain-containing proteins have so far been shown to demonstrably have Rab/GAP activity. The human EVI5 protein, which was identified at the breakpoint in a constitutional chromosome translocation in a patient with stage 4S neuroblastoma (Roberts et al., 1998), contains a TBC domain near its N terminus. EVI5 has been identified in the centrosome in interphase cells (Faitar et al., 2005) and in the midbody during the terminal stages of cytokinesis. Small interfering RNA knockdown of EVI5 results in multinucleate cells because of an inability of daughter cell abscission (Faitar et al., 2006). Because of the essential role of EVI5 in cytokinesis, as well as its implicated involvement in cancer development, we used a proteomics approach and identified the Rab protein that is activated by the EVI5 TBC domain.Rab proteins function by cycling between the biologically active GTP-bound form and the guanosine diphosphate (GDP)-bound inactive form. In the active GTP-bound conformation, these proteins can directly interact with specific eff...

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