Bruce A. Knutson

Mechanism of Mediator Recruitment by Tandem Gcn4 Activation Domains and Three Gal11 Activator-Binding Domains

ABSTRACT Targets of the tandem Gcn4 acidic activation domains in transcription preinitiation complexes were identified by site-specific cross-linking. The individual Gcn4 activation domains cross-link to three common targets, Gal11/Med15, Taf12, and Tra1, which are subunits of four conserved coactivator complexes, Mediator, SAGA, TFIID, and NuA4. The Gcn4 N-terminal activation domain also cross-links to the Mediator subunit Sin4/Med16. The contribution of the two Gcn4 activation domains to transcription was gene specific and varied from synergistic to less than additive. Gcn4-dependent genes had a requirement for Gal11 ranging from 10-fold dependence to complete Gal11 independence, while the Gcn4-Taf12 interaction did not significantly contribute to the expression of any gene studied. Complementary methods identified three conserved Gal11 activator-binding domains that bind each Gcn4 activation domain with micromolar affinity. These Gal11 activator-binding domains contribute additively to transcription activation and Mediator recruitment at Gcn4- and Gal11-dependent genes. Although we found that the conserved Gal11 KIX domain contributes to Gal11 function, we found no evidence of specific Gcn4-KIX interaction and conclude that the Gal11 KIX domain does not function by specific interaction with Gcn4. Our combined results show gene-specific coactivator requirements, a surprising redundancy in activator-target interactions, and an activator-coactivator interaction mediated by multiple low-affinity protein-protein interactions.

Yeast Rrn7 and Human TAF1B Are TFIIB-Related RNA Polymerase I General Transcription Factors

RNA polymerase I uses a transcription factor IIB–related protein for transcription, similar to the known requirement for polymerase II and III. Eukaryotic and archaeal multisubunit RNA polymerases (Pols) are structurally related and require several similar components for transcription initiation. However, none of the Pol I factors were known to share homology with transcription factor IIB (TFIIB) or TFIIB-related proteins, key factors in the initiation mechanisms of the other Pols. Here we show that Rrn7, a subunit of the yeast Pol I core factor, and its human ortholog TAF1B are TFIIB-like factors. Although distantly related, Rrn7 shares many activities associated with TFIIB-like factors. Domain swaps between TFIIB-related factors show that Rrn7 is most closely related to the Pol III general factor Brf1. Our results point to the conservation of initiation mechanisms among multisubunit Pols and reveal a key function of yeast core factor/human SL1 in Pol I transcription.

Domains of Tra1 important for activator recruitment and transcription coactivator functions of SAGA and NuA4 complexes.

ABSTRACT The Tra1 protein is a direct transcription activator target that is essential for coactivator function of both the SAGA and NuA4 histone acetyltransferase (HAT) complexes. The ∼400-kDa Saccharomyces cerevisiae Tra1 polypeptide and its human counterpart TRRAP contain 67 or 68 tandem α-helical HEAT and TPR protein repeats that extend from the N terminus to the conserved yet catalytically inactive phosphatidylinositol 3-kinase (PI3K) domain. We generated a series of mutations spanning the length of the protein and assayed for defects in transcription, coactivator recruitment, and histone acetylation at SAGA- and NuA4-dependent genes. Inviable TRA1 mutants all showed defects in SAGA and NuA4 complex stability, suggesting that similar surfaces of Tra1 mediate assembly of these two very different coactivator complexes. Nearly all of the viable Tra1 mutants showed transcription defects that fell into one of three classes: (i) defective recruitment to promoters, (ii) reduced stability of the SAGA and NuA4 HAT modules, or (iii) normal recruitment of Tra1-associated subunits but reduced HAT activity in vivo. Our results show that Tra1 recruitment at Gcn4-dependent and Rap1-dependent promoters requires the same regions of Tra1 and that separate regions of Tra1 contribute to the HAT activity and stability of the SAGA and NuA4 HAT modules.

Architecture of the Saccharomyces cerevisiae RNA polymerase I Core Factor complex

Core Factor (CF) is a conserved RNA polymerase (Pol) I general transcription factor comprising Rrn6, Rrn11 and the TFIIB-related subunit Rrn7. CF binds TATA-binding protein (TBP), Pol I and the regulatory factors Rrn3 and upstream activation factor. We used chemical cross-linking–MS to determine the molecular architecture of CF and its interactions with TBP. The CF subunits assemble through an interconnected network of interactions between five structural domains that are conserved in orthologous subunits of the human Pol I factor SL1. We validated the cross-linking–derived model through a series of genetic and biochemical assays. Our combined results show the architecture of CF and the functions of the CF subunits in assembly of the complex. We extend these findings to model how CF assembles into the Pol I preinitiation complex, providing new insight into the roles of CF, TBP and Rrn3.

Insights into the domain and repeat architecture of target of rapamycin

A simple and efficient protein sequence analysis strategy was developed to predict the number and location of structural repeats in the TOR protein. This strategy uses multiple HHpred alignments against proteins of known 3D structure to enable protein repeats referenced from the 3D structure to be traced back to the query protein sequence by using user-directed repeat assignments. The HHpred strategy performed with high sensitivity by predicting 100% of the repeat units within a test set of HEAT- and TPR-repeat-containing proteins of known three-dimensional structure. The HHpred strategy predicts that TOR contains 32 tandem HEAT repeats extending from the N-terminus to the FAT domain, which is itself comprised of 16 tandem TPR repeats. These findings were used to assemble a 3D atomic model for the TOR protein.

TFIIB-related factors in RNA polymerase I transcription.

Eukaryotic RNA polymerases (Pol) I, II, III and archaeal Pol use a related set of general transcription factors to recognize promoter sequences and recruit Pol to promoters and to function at key points in the transcription initiation mechanism. The TFIIB-like general transcription factors (GTFs) function during several important and conserved steps in the initiation pathway for Pols II, III, and archaeal Pol. Until recently, the mechanism of Pol I initiation seemed unique, since it appeared to lack a GTF paralogous to the TFIIB-like proteins. The surprising recent discovery of TFIIB-related Pol I general factors in yeast and humans highlights the evolutionary conservation of transcription initiation mechanisms for all eukaryotic and archaeal Pols. These findings reveal new roles for the function of the Pol I GTFs and insight into the function of TFIIB-related factors. Models for Pol I transcription initiation are reexamined in light of these recent findings. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Structural mechanism of ATP-independent transcription initiation by RNA polymerase I

Transcription initiation by RNA Polymerase I (Pol I) depends on the Core Factor (CF) complex to recognize the upstream promoter and assemble into a Pre-Initiation Complex (PIC). Here, we solve a structure of Saccharomyces cerevisiae Pol I-CF-DNA to 3.8 Å resolution using single-particle cryo-electron microscopy. The structure reveals a bipartite architecture of Core Factor and its recognition of the promoter from −27 to −16. Core Factor’s intrinsic mobility correlates well with different conformational states of the Pol I cleft, in addition to the stabilization of either Rrn7 N-terminal domain near Pol I wall or the tandem winged helix domain of A49 at a partially overlapping location. Comparison of the three states in this study with the Pol II system suggests that a ratchet motion of the Core Factor-DNA sub-complex at upstream facilitates promoter melting in an ATP-independent manner, distinct from a DNA translocase actively threading the downstream DNA in the Pol II PIC. DOI: http://dx.doi.org/10.7554/eLife.27414.001

Emergence and expansion of TFIIB-like factors in the plant kingdom.

Many gene families in higher plants have expanded in number, giving rise to diverse protein paralogs with specialized biochemical functions. For instance, plant general transcription factors such as TFIIB have expanded in number and in some cases perform specialized transcriptional functions in the plant cell. To date, no comprehensive genome-wide identification of the TFIIB gene family has been conducted in the plant kingdom. To determine the extent of TFIIB expansion in plants, I used the remote homology program HHPred to search for TFIIB homologs in the plant kingdom and performed a comprehensive analysis of eukaryotic TFIIB gene families. I discovered that higher plants encode more than 10 different TFIIB-like proteins. In particular, Arabidopsis thaliana encodes 14 different TFIIB-like proteins and predicted domain architectures of the newly identified TFIIB-like proteins revealed that they have unique modular domain structures that are divergent in sequence and size. Phylogenetic analysis of selected eukaryotic organisms showed that most life forms encode three major TFIIB subfamilies that include TFIIB, Brf, Rrn7/TAF1B/MEE12 subfamilies, while all plants and some algae species encode one or two additional TFIIB-related protein subfamilies. A subset of A. thaliana GTFs have also expanded in number, indicating that GTF diversification and expansion is a general phenomenon in higher plants. Together, these findings were used to generate a model for the evolutionary history of TFIIB-like proteins in eukaryotes.

Treacher Collins syndrome mutations in Saccharomyces cerevisiae destabilize RNA polymerase I and III complex integrity

Treacher Collins syndrome (TCS) is a craniofacial disorder that is characterized by the malformation of the facial bones. Mutations in three genes (TCOF1, POLR1C and POLR1D) involved in RNA polymerase I (Pol I) transcription account for more than 90% of disease cases. Two of these TCS-associated genes, POLR1C and POLR1D, encode for essential Pol I/III subunits that form a heterodimer necessary for Pol I/III assembly, and many TCS mutations lie along their evolutionarily conserved dimerization interface. Here we elucidate the molecular basis of TCS mutations in Saccharomyces cerevisiae, and present a new model for how TCS mutations may disrupt Pol I and III complex integrity.

Breaking the mold: structures of the RNA polymerase I transcription complex reveal a new path for initiation

ABSTRACT While structures of the RNA polymerase (Pol) II initiation complex have been resolved and extensively studied, the Pol I initiation complex remained elusive. Here, we review the recent structural analyses of the yeast Pol I transcription initiation complex that reveal several unique and unexpected Pol I-specific properties.