The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a “fuzzy complex” where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets.

The Saccharomyces cerevisiae kinase Bur1 is involved in coupling transcription elongation to chromatin modification, but not all important Bur1 targets in the elongation complex are known. Using a chemical genetics strategy wherein Bur1 kinase was engineered to be regulated by a specific inhibitor, we found that Bur1 phosphorylates the Spt5 C-terminal repeat domain (CTD) both in vivo and in isolated elongation complexes in vitro. Deletion of the Spt5 CTD or mutation of the Spt5 serines targeted by Bur1 reduces recruitment of the PAF complex, which functions to recruit factors involved in chromatin modification and mRNA maturation to elongating polymerase II (Pol II). Deletion of the Spt5 CTD showed the same defect in PAF recruitment as rapid inhibition of Bur1 kinase activity, and this Spt5 mutation led to a decrease in histone H3K4 trimethylation. Brief inhibition of Bur1 kinase activity in vivo also led to a significant decrease in phosphorylation of the Pol II CTD at Ser-2, showing that Bur1 also contributes to Pol II Ser-2 phosphorylation. Genetic results suggest that Bur1 is essential for growth because it targets multiple factors that play distinct roles in transcription.

SUMMARY Previous studies suggested that expression of most yeast mRNAs is dominated by either transcription factor TFIID or SAGA. We reexamined the role of TFIID by rapid depletion of S. cerevisiae TFIID subunits and measurement of changes in nascent transcription. We find that transcription of nearly all mRNAs is strongly dependent on TFIID function. Degron-dependent depletion of Tafs 1,2,7,11, and 13 showed similar transcription decreases for genes in the Taf1-depleted, Taf1-enriched, TATA-containing and TATA-less gene classes. The magnitude of TFIID-dependence varies with growth conditions, although this variation is similar genome-wide. Many studies have suggested differences in gene regulatory mechanisms between TATA and TATA-less genes and these differences have been attributed in part to differential dependence on SAGA or TFIID. Our work indicates that TFIID participates in expression of nearly all yeast mRNAs and that differences in regulation between these two gene categories is due to other properties.

The non-natural photoreactive amino acid p-Benzoyl-L-Phenylalanine (Bpa) was incorporated into the RNA polymerase (Pol) II surface surrounding the central cleft formed by the Rpb1 and Rpb2 subunits. Photocrosslinking of Preinitiation Complexes (PICs) with these Pol II derivatives and hydroxyl radical cleavage assays revealed that the TFIIF dimerization domain interacts with the Rpb2 lobe and protrusion domains adjacent to Rpb9 while TFIIE crosslinks to the Rpb1 clamp domain on the opposite side of the Pol II central cleft. Mutations in the Rpb2 lobe and protrusion domains were found to alter both Pol II-TFIIF binding and the transcription start site, a phenotype associated with mutations in TFIIF, Rpb9, and TFIIB. In combination with previous biochemical and structural studies, these new findings illuminate the structural organization of the PIC and reveal a network of protein-protein interactions involved in transcription start site selection.The earliest step in transcription initiation is recruitment of Pol II and the general transcription factors to form the Preinitiation Complex (PIC) 1 . Upon addition of ATP, the PIC transitions to the Open Complex state, resulting in separation of the DNA strands surrounding the transcription start site and insertion of the template strand into the active center of Pol II. Next, Pol II must locate the transcription start site, which in S. cerevisiae, can be over 60 base pairs distant from the initial site of DNA strand separation 2 . Mutations in the general factor TFIIF and in the B-finger domain of the general factor TFIIB can alter the transcription start site, suggesting these two factors are involved in start site recognition 3-7 . Consistent with this role, crystallography and protein-protein crosslinking in the PIC have positioned both of these factors within the Pol II active site cleft 8-10 . Additionally, mutations in the Pol II subunit Rpb9 have been found to alter the transcription start site, but it remains unclear how this subunit, which is located distant from the Pol II active site, participates in start site selection 11,12 .Initiation of RNA synthesis begins with DNA-NTP base pairing, phosphodiester bond formation, and translocation of the DNA-RNA hybrid within the active center of Pol II. After synthesis of 8−12 bases of RNA, Pol II escapes from the promoter into a stable elongation state 13-15 . The nucleation of transcription factors at the promoter and the structural transition into the open and elongation complexes involves a complex set of protein-protein and protein-DNA interactions. Elucidating these molecular interactions within the transcription machinery Phone: 206 667 5261 Fax 206 667 6497 shahn@fhcrc.org. AUTHOR CONTRIBUTIONS L.W. modified the non-natural amino acid incorporation system and performed the experiment in Figure 1. H-T.C. performed and designed the remaining experiments. S.H. supervised the study. H-T.C. and S.H. wrote the manuscript.Publisher's Disclaimer: This PDF receipt will only be used as the basis for generating ...

Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a "fuzzy" protein interface. In contrast to Gcn4, the activity of these synthetic ADs is not strongly dependent on any one residue of the AD, and this redundancy is similar to that of some natural ADs in which few if any sequence-specific residues have been identified. The additional hydrophobic residues in the synthetic ADs likely allow multiple faces of the AD helix to interact with the Gal11 activator-binding domain, effectively forming a fuzzier interface than that of the wild-type cAD.Mediator complex | protein NMR T ranscription activators are regulators of cell identity, cell growth, and the response to environmental conditions. These highly regulated factors contain one or more activation domains (ADs) that typically bind coactivators-the complexes that contact the transcription machinery and/or have chromatin modifying activity (1-5). AD-coactivator binding initiates a cascade of events leading to productive transcription including targeted chromatin remodeling and stimulation of both RNA polymerase II preinitiation complex formation and transcription elongation (6). Many broadly acting ADs bind several coactivators, allowing them to function at a wide range of promoters with different coactivator requirements (7)(8)(9)(10)(11)(12)(13)(14). The function of most tested ADs is conserved among eukaryotes (15, 16), even though some key activator targets are not conserved. For example, the herpes virus protein VP16 strongly activates transcription in both yeast and mammalian cells, although human Med25, a critical target of VP16 in humans, is not found in yeast (17,18). Thus, the ability to adapt to different coactivator targets is seemingly a key property of ADs.Defining what constitutes a functional AD has been difficult, because there is little apparent sequence similarity among different ADs. All structurally characterized eukaryotic ADs lack a stable 3D structure and are disordered in the absence of a coactivator target (19)(20)(21)(22)(23)(24)(25). Initial studies classified ADs based on enriched residues: acidic proline-, glutamine-, or serine-rich activators (26). However, these residue types later were found to be generally enriched in intrinsically disordered proteins and were termed "disorder-promoting residues" (27, 28).Attempts to define functional sequence motifs within ADs have led to ambiguous results. For many ...