Transcriptional activation by artificial recruitment in yeast is influenced by promoter architecture and downstream sequences.

The idea that recruitment of the transcriptional machinery to a promoter suffices for gene activation is based partly on the results of "artificial recruitment" experiments performed in vivo. Artificial recruitment can be effected by a "nonclassical" activator comprising a DNA-binding domain fused to a component of the transcriptional machinery. Here we show that activation by artificial recruitment in yeast can be sensitive to any of three factors: position of the activator-binding elements, sequence of the promoter, and coding sequences downstream of the promoter. In contrast, classical activators worked efficiently at all promoters tested. In all cases the "artificial recruitment" fusions synergized well with classical activators. A classical activator evidently differs from a nonclassical activator in that the former can touch multiple sites on the transcriptional machinery, and we propose that that difference accounts for the broader spectrum of activity of the typical classical activator. A similar conclusion is reached from studies in mammalian cells in the accompanying paper [Nevado, J., Gaudreau, L., Adam, M. & Ptashne, M. (1999) Proc. Natl. Acad. Sci. USA 96, 2674-2677].

Polymerase (Pol) III TATA box-binding protein (TBP)-associated factor Brf binds to a surface on TBP also required for activated Pol II transcription.

The TATA box-binding protein (TBP) plays an essential role in transcription by all three eukaryotic nuclear RNA polymerases, polymerases (Pol) I, II, and III. In each case, TBP interacts with class-specific TBP-associated factors (TAFs) to form class-specific transcription initiation factors. For yeast Pol III transcription, TBP associates with Brf (from TFIIB-related factor) and B", two Pol III TAFs, to form Pol III transcription factor TFIIIB. Here, we identify TBP surface residues that are required for interaction with yeast Pol III TAFs. Ninety-one human TBP surface residue mutants with radical substitutions were analyzed for the ability to form stable gel shift complexes with purified Brf and B" and for their activities for in vitro synthesis of yeast U6 snRNA. Mutations in a large positively charged epitope extending from the top (that is, on the surface opposite the DNA-facing "saddle" of TBP) and onto the side of the first TBP repeat inhibited binding to Brf (residues K181, L185, R186, E206, R231, L232, R235, K236, R239, Q242, K243, K249, and F250). A triple-mutant TBP (R231E + R235E + R239S) had greatly reduced activity for yeast U6 snRNA gene transcription while remaining active for Pol II basal transcription. Similar results were observed when selected mutations were introduced into yeast TBP at equivalent positions. A C-terminal fragment of Brf lacking the region of homology with TFIIB retains the ability to bind TBP-DNA complexes (G. Kassavetis, C. Bardeleben, A. Kumar, E. Ramirez, and E. P. Geiduschek, Mol. Cell. Biol. 17:5299-5306, 1997); the same TBP mutations reduced binding by this fragment. Mutations in TBP residues that interact with TFIIB did not affect Brf binding or U6 gene transcription. These results indicate that Brf and TFIIB interact differently with TBP. An extensively overlapping epitope on the top surface of TBP was found previously to be required for activated Pol II transcription and has been hypothesized to interact with Pol II TAFs. Our results map the surface of TBP that interacts with Brf and suggest that Pol II and Pol III TAFs interact with the same surface of TBP.

Radical mutations reveal TATA-box binding protein surfaces required for activated transcription in vivo.

Regions on the surface of human TATA-box binding protein (TBP) required for activated transcription in vivo were defined by construction of a library of 89 surface residue mutants with radical substitutions that were assayed for their ability to support activated transcription in vivo, basal transcription in vitro, and TFIIA and TFIIB binding in vitro. Four epitopes were identified in which substitutions in two to four neighboring surface residues greatly inhibited activated transcription in vivo. One epitope in which substitutions inhibited both basal and activated transcription (E284, L287) is the interface between TBP and TFIIB. Another (A184, N189, E191, R205) is the recently determined interface between TBP and TFIIA. Mutations in residues in this TFIIA interface greatly inhibit activated, but not basal transcription, demonstrating a requirement for the TFIIA-TBP interaction for activated transcription in vivo in mammalian cells. The remaining two activation epitopes (TBP helix 2 residues R231, R235, R239, plus F250; and G175, C176, P247) are probably interfaces with other proteins required for activated transcription. The library of mutants responded virtually identically to two different types of activators, GL4-E1A and GAL4-VP16, indicating that transcriptional activation by different classes of activators requires common interactions with TBP.

Cloning of the YenI restriction endonuclease and methyltransferase from Yersinia enterocolitica serotype O8 and construction of a transformable R-M+ mutant.

Two different clonal groups of pathogenic Yersinia enterocolitica strains, American and non-American, have been recognized. These are distinguished by a number of criteria, including their virulence in a murine model of infection. However, genetic analysis of virulence in American strains has been hampered due to the severe restriction of transformed or electroporated DNA. Thus, we cloned the yenIMR locus from the American serotype strain 8081c, which encodes YenI, an isoschizomer of PstI. This clone encodes both the restriction endonuclease and methyltransferase. The location of the genes on the clone was determined and this information was used to construct a small deletion (400 bp) that results in an R-M+ phenotype. This mutation was recombined onto the Y. enterocolitica chromosome to give an R-M+ mutant which showed at least a 1000-fold increase in electroporation frequency compared to the wild-type strain. Southern analysis using a probe derived from yenIMR indicated that American serotype strains have this locus whereas non-American serotype strains do not.

Adenovirus E1A activation domain binds the basic repeat in the TATA box transcription factor.

The adenovirus large E1A protein is a potent activator of transcription. We use several different experimental approaches to demonstrate that the large E1A protein binds specifically and stably to the TATA box-binding factor (TFIID), the general polymerase II transcription factor that initiates assembly of transcription complexes. Sedimentation velocity centrifugation revealed that TFIID and E1A form a heterodimer in vitro. We demonstrate that the activation domain of E1A (conserved region 3) binds to TFIID. E1A interacts with a 51 residue region from the conserved C-terminal domain of TFIID that includes a repeat of basic residues between the homologous direct repeats of TFIID. Analysis of TFIID binding by various E1A mutants indicates that TFIID binding is necessary, although not sufficient, for E1A transactivation.