The activities of several mRNA processing factors are coupled to transcription through binding to RNA polymerase II (Pol II). The largest subunit of Pol II contains a repetitive carboxy-terminal domain (CTD) that becomes highly phosphorylated during transcription. mRNA-capping enzyme binds only to phosphorylated CTD, whereas other processing factors may bind to both phosphorylated and unphosphorylated forms. Capping occurs soon after transcription initiation and before other processing events, raising the question of whether capping components remain associated with the transcription complex after they have modified the 5 end of the mRNA. Chromatin immunoprecipitation in Saccharomyces cerevisiae shows that capping enzyme cross-links to promoters but not coding regions. In contrast, the mRNA cap methyltransferase and the Hrp1/CFIB polyadenylation factor cross-link to both promoter and coding regions. Remarkably, the phosphorylation pattern of the CTD changes during transcription. Ser 5 phosphorylation is detected primarily at promoter regions dependent on TFIIH. In contrast, Ser 2 phosphorylation is seen only in coding regions. These results suggest a dynamic association of mRNA processing factors with differently modified forms of the polymerase throughout the transcription cycle. Eukaryotic mRNAs undergo 5Ј end capping, splicing of introns, and polyadenylation. Targeting of capping enzyme and other RNA processing factors is through binding to the carboxy-terminal domain (CTD) of the RNA polymerase II (Pol II) largest subunit McCracken et al. 1997a,b;Yue et al. 1997;Hirose and Manley 1998;Pillutla et al. 1998;Hirose et al. 1999). Capping is the earliest modification, occurring when the transcript is 20 to 40 nucleotides long (Jove and Manley 1984;Rasmussen and Lis 1993). Phosphorylation of the CTD occurs soon after initiation and is necessary for capping enzyme recruitment. Other RNA-processing factors bind to both phosphorylated and unphosphorylated CTD and act much later during transcription. This raises the question of whether capping enzyme and other processing factors are simultaneously associated with RNA pol II throughout transcription or instead interact transiently at different stages. In vivo cross-linking is used here to show that capping enzyme is recruited to promoter regions dependent on TFIIH kinase activity, but does not remain associated with elongating polymerase. In contrast, the mRNA cap methyltransferase Abd1 and the polyadenylation factor CFIB/Hrp1 cross-link throughout transcribed regions. Surprisingly, Ser 5 phosphorylation of the CTD also localizes to promoters, suggesting dephosphorylation not long after escape into elongation phase. Ser 2 phosphorylation of the CTD shows a complementary pattern, with no cross-linking at the promoter and higher levels near the 3Ј end of the gene. These results suggest a dynamic association of RNA processing factors with differently modified forms of the polymerase throughout the transcription cycle. Results Experimental designTo determine the in vivo ...
In yeast, the TBP-associated factors (TAFs) Taf17, Taf60, and Taf61(68) resemble histones H3, H4, and H2B, respectively. To analyze their roles in vivo, conditional alleles were isolated by mutagenizing their histone homology domains. Conditional alleles of TAF17 or TAF60 can be specifically suppressed by overexpression of any of the other histone-like TAFs. This and other genetic evidence supports the model of a histone octamer-like structure within TFIID. Shifting strains carrying the conditional TAF alleles to non-permissive conditions results in degradation of TFIID components and the rapid loss of mRNA production. Therefore, in contrast to previous studies in yeast that found only limited roles for TAFs in transcription, we find that the histone-like TAFs are generally required for in vivo transcription.
Introduction: DLL3, an atypical Notch ligand, is expressed in SCLC tumors but is not detectable in normal adult tissues. Rovalpituzumab tesirine (Rova-T) is an antibody-drug conjugate containing a DLL3targeting antibody tethered to a cytotoxic agent pyrrolobenzodiazepine by means of a protease-cleavable linker. The efficacy and safety of Rova-T compared with topotecan as second-line therapy in patients with SCLC expressing high levels of DLL3 (DLL3-high) was evaluated.Methods: The TAHOE study was an open-label, two-to-one randomized, phase 3 study comparing Rova-T with topotecan as second-line therapy in DLL3-high advanced or metastatic SCLC. Rova-T (0.3 mg/kg) was administered intravenously on day 1 of a 42-day cycle for two cycles, with two additional cycles available to patients who met protocol-defined criteria for continued dosing. Topotecan (1.5 mg/m 2 ) was administered intravenously on days 1 to 5 of a 21-day cycle. The primary end point was overall survival (OS).Results: Patients randomized to Rova-T (n ¼ 296) and topotecan (n ¼ 148) were included in the efficacy analyses. The median age was 64 years, and 77% had the extensive disease at initial diagnosis. The median OS (95% confidence interval) was 6.3 months (5.6-7.3) in the Rova-T arm and 8.6 months (7.7-10.1) in the topotecan arm (hazard ratio, 1.46 [95% confidence interval: 1.17-1.82]). An independent data monitoring committee recommended that enrollment be discontinued because of the shorter OS observed with Rova-T compared with topotecan. Safety profiles for both drugs were consistent with previous reports.Conclusions: Compared with topotecan, which is the current standard second-line chemotherapy, Rova-T exhibited an inferior OS and higher rates of serosal effusions, photosensitivity reaction, and peripheral edema in patients with SCLC. A considerable unmet therapeutic need remains in this population.
The yeast transcriptional activator ADR1, which is required for ADH2 and peroxisomal gene expression, contains four separable and partially redundant activation domains (TADs). Mutations in ADA2 or GCN5, encoding components of the ADA coactivator complex involved in histone acetylation, severely reduced LexA-ADR1-TAD activation of a LexA-lacZ reporter gene. Similarly, the ability of the wild-type ADR1 gene to activate an ADH2-driven promoter was compromised in strains deleted for ADA2 or GCN5. In contrast, defects in other general transcription cofactors such as CCR4, CAF1/POP2, and SNF/SWI displayed much less or no effect on LexA-ADR1-TAD activation. Using an in vitro protein binding assay, ADA2 and GCN5 were found to specifically contact individual ADR1 TADs. ADA2 could bind TAD II, and GCN5 physically interacted with all four TADs. Both TADs I and IV were also shown to make specific contacts to the C-terminal segment of TFIIB. In contrast, no significant binding to TBP was observed. TAD IV deletion analysis indicated that its ability to bind GCN5 and TFIIB was directly correlated with its ability to activate transcription in vivo. ADR1 TADs appear to make several contacts, which may help explain both their partial redundancy and their varying requirements at different promoters. The contact to and dependence on GCN5, a histone acetyltransferase, suggests that rearrangement of nucleosomes may be one important means by which ADR1 activates transcription.In Saccharomyces cerevisiae, the transcriptional activator ADR1 is required for expression of the glucose-repressible alcohol dehydrogenase gene (ADH2) under nonfermentative conditions (1). It also regulates genes required in glycerol metabolism (2, 3) and peroxisome function and biogenesis (4, 5). ADR1 is a zinc finger, DNA-binding protein that is 153 kDa in size (6, 7). Its regulation of ADH2 under nonfermentative growth conditions occurs by binding to UAS1, a palindromic site, located 110 bp 1 upstream of the ADH2 TATAA sequence The presence of four transactivation regions suggests that ADR1 may make multiple protein contacts to transcriptional cofactors and/or core transcriptional components. The observation that TADs II and III are functionally redundant (9) suggests that some of these contacts may be made to the same protein.There are a number of potential targets for ADR1 activation domains. Core transcriptional components including TBP, TFIIB, TFIIF, TFIIE, and TAFs have been implicated in mammalians system as being direct contacts for transcriptional activators (12). In yeast, the GAL4 activation domain has been shown to bind TBP but not TFIIB in vitro (13). In addition to these core transcriptional factors, other cofactors or coactivators may mediate the action of activators. The ADA2 complex is one such coactivator complex. These proteins have been shown to bind activators like VP16 and GCN4 (14, 15) and to be required for maximal transcriptional activity of several yeast activators (16). However, some yeast activators like HAP4 and GAL4 (16,17) are ...
Many questions remain concerning the role of TFIID TBP-associated factors (TAFs) in transcription, including whether TAFs are required at most or only a small subset of promoters. It was shown previously that three histone-like TAFs are broadly required for transcription, but interpretation of this observation is complicated because these proteins are components of both TFIID and the SAGA histone acetyltransferase complex. Here we show that mutations in the yeast TFIID-specific protein Taf40 lead to a general cessation of transcription, even in the presence of excess TBP, suggesting that the TFIID complex is required at most promoters in vivo. Received May 6, 1999; revised version accepted August 11, 1999. RNA polymerase II (Pol II) is positioned on a promoter by a set of accessory basal transcription factors. One of these factors, TFIID, binds the consensus promoter sequence TATAA and nucleates the assembly of other transcription factors. One subunit of TFIID, the TATAbinding protein (TBP), is necessary and sufficient for this activity in vitro. However, TBP in vivo is associated with several multisubunit complexes that mediate transcription by RNA polymerase I, II, or III (Hernandez 1993). The TBP-containing complexes implicated in Pol II transcription are TFIID, the TBP-MOT1 complex (Auble and Hahn 1993), and the SNAPc complex in higher eukaryotes (Hernandez 1993). In addition, the SAGA histone acetyltransferase complex can associate with TBP transiently (Eisenmann et al. 1992;Sterner et al. 1999).The TFIID complex consists of TBP and 10-12 TBPassociated factors (TAFs). The essential role of TBP in Pol II-mediated transcription has been demonstrated both in vivo and in vitro (Hampsey 1998). The functions of TAFs in transcription are less clear (for review, see Verrijzer and Tjian 1996;Hoffmann et al. 1997;Hahn 1998). In vitro experiments in mammalian, Drosophila, or yeast systems have indicated a requirement for TAFs in responding to transcriptional activators but not for basal transcription (Kokubo et al. 1993;Chen et al. 1994;Reese et al. 1994). However, activated transcription in vitro has also been reported in a yeast system apparently lacking TAFs, with activation being mediated by the Pol II-associated mediator/SRB proteins (Kim et al. 1994;Koleske and Young 1994). TAF-independent activation has also been reported in mammalian in vitro transcription systems (Oelgeschlager et al. 1998;Wu et al. 1998). Therefore, there may be both TAF-dependent and -independent activation mechanisms that can be emphasized by the particular choice of in vitro transcription system. In vivo experiments are therefore essential for testing the physiological relevance of in vitro results.Several other roles for TAFs have been documented in addition to their proposed function as transcriptional coactivators. TFIID makes extensive contacts with core promoter elements in addition to the TATA element, and these contacts are made by the TAFs (for review, see Hoffmann et al. 1997). Furthermore, the largest TAF can inhibit TBP bin...
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