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TFIID TAF6-TAF9 complex formation involves the HEAT repeat-containing C-terminal domain of TAF6 and is modulated by TAF5 protein.

ABSTRACT: The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes.

SUBMITTER: Scheer E

PROVIDER: S-EPMC3431708 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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TFIID TAF6-TAF9 complex formation involves the HEAT repeat-containing C-terminal domain of TAF6 and is modulated by TAF5 protein.

Scheer Elisabeth E Delbac Frédéric F Tora Laszlo L Moras Dino D Romier Christophe C

The Journal of biological chemistry 20120613 33

The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved ...[more]

PMID: 22696218

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Project description:TFIID and SAGA complexes play a critical role in RNA Polymerase II dependent activated transcription. Although the two regulatory complexes are recruited to promoters by activation domain-interactions, the contribution of the different subunits or the different domains of the individual subunits is not completely understood. Taf9 is a shared subunit in TFIID and SAGA and has an N-terminal H3-like histone fold domain and a highly conserved C-terminal domain, Taf9-CTD. In this study, we have uncovered an essential role for the Taf9-CTD in transcriptional activation. The Taf9-CTD was not essential for the histone-fold mediated interaction with Taf6, SAGA and TFIID integrity or Gcn4 interaction with SAGA. Transcriptome profiling performed under Gcn4 activating conditions showed that the Taf9-CTD is required for expression of ~17% of the yeast genome and provides a coactivator function to recruit TFIID and SAGA complexes to the promoters in vivo during transcriptional activation. Integrated genome-wide data analysis showed that the Taf9-CTD is required for activation of promoters bound by several transcription factors indicating a broad role for Taf9-CTD in promoter occupancy of TFIID or SAGA complexes. Interestingly, only a subset of the promoters seemed to be dependent on the Taf9-CTD for assembly of the pre-initiation complex indicating redundancy in activator targets to assemble PIC in vivo. Together these results indicate that evolutionarily conserved domains in shared subunits of TFIID and SAGA have a pervasive role in genome-wide transcription. This GEO series consists of 14 microarray hybridizations using the Agilent two-color experiment with the Agilent Custom Saccharomyces cerevisiae 8x15k gene expression array. Four biological replicates each for the wild-type (TAF9), the mutant taf9-tCRD2 strain treated or untreated with SM, and the wild-type (TAF9) versus mutant taf9-tCRD2 treated with SM hybridized as dye-swapped replicates. Two biological replicates for wild-type (SPT20) vs spt20D strains treated with SM, and hybridized as dye-swapped replicates to identify the fraction of SAGA dependent genes under amino-acid starvation conditions. The overall aim was to identify genes dependent on the conserved C-terminal region domain of TAF9 and determine their dependence on the SAGA subunit Spt20 for expression.

Project description:UnlabelledFimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions.ImportanceFimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

Dataset Information

TFIID TAF6-TAF9 complex formation involves the HEAT repeat-containing C-terminal domain of TAF6 and is modulated by TAF5 protein.

Publications

TFIID TAF6-TAF9 complex formation involves the HEAT repeat-containing C-terminal domain of TAF6 and is modulated by TAF5 protein.

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