Core Domain Of TFIIB Research Articles

Structural dissection of an interaction between transcription initiation and termination factors implicated in promoter-terminator cross-talk

Functional cross-talk between the promoter and terminator of a gene has long been noted. Promoters and terminators are juxtaposed to form gene loops in several organisms, and gene looping is thought to be involved in transcriptional regulation. The general transcription factor IIB (TFIIB) and the C-terminal domain phosphatase Ssu72, essential factors of the transcription preinitiation complex and the mRNA processing and polyadenylation complex, respectively, are important for gene loop formation. TFIIB and Ssu72 interact both genetically and physically, but the molecular basis of this interaction is not known. Here we present a crystal structure of the core domain of TFIIB in two new conformations that differ in the relative distance and orientation of the two cyclin-like domains. The observed extraordinary conformational plasticity may underlie the binding of TFIIB to multiple transcription factors and promoter DNAs that occurs in distinct stages of transcription, including initiation, reinitiation, and gene looping. We mapped the binding interface of the TFIIB-Ssu72 complex using a series of systematic, structure-guided in vitro binding and site-specific photocross-linking assays. Our results indicate that Ssu72 competes with acidic activators for TFIIB binding and that Ssu72 disrupts an intramolecular TFIIB complex known to impede transcription initiation. We also show that the TFIIB-binding site on Ssu72 overlaps with the binding site of symplekin, a component of the mRNA processing and polyadenylation complex. We propose a hand-off model in which Ssu72 mediates a conformational transition in TFIIB, accounting for the role of Ssu72 in transcription reinitiation, gene looping, and promoter-terminator cross-talk.

Dynamic Architecture of a Minimal RNA Polymerase II Open Promoter Complex

The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ single-molecule Förster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture ofa minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.

Architecture of the Yeast RNA Polymerase II Open Complex and Regulation of Activity by TFIIF

To investigate the function and architecture of the open complex state of RNA polymerase II (Pol II), Saccharomyces cerevisiae minimal open complexes were assembled by using a series of heteroduplex HIS4 promoters, TATA binding protein (TBP), TFIIB, and Pol II. The yeast system demonstrates great flexibility in the position of active open complexes, spanning 30 to 80 bp downstream from TATA, consistent with the transcription start site scanning behavior of yeast Pol II. TFIIF unexpectedly modulates the activity of the open complexes, either repressing or stimulating initiation. The response to TFIIF was dependent on the sequence of the template strand within the single-stranded bubble. Mutations in the TFIIB reader and linker region, which were inactive on duplex DNA, were suppressed by the heteroduplex templates, showing that a major function of the TFIIB reader and linker is in the initiation or stabilization of single-stranded DNA. Probing of the architecture of the minimal open complexes with TFIIB-FeBABE [TFIIB-p-bromoacetamidobenzyl-EDTA-iron(III)] derivatives showed that the TFIIB core domain is surprisingly positioned away from Pol II, and the addition of TFIIF repositions the TFIIB core domain to the Pol II wall domain. Together, our results show an unexpected architecture of minimal open complexes and the regulation of activity by TFIIF and the TFIIB core domain.

Dynamic Architecture of the RNA Polymerase II Open Promoter Complex

Transcription initiation of eukaryotic protein-coding genes is a multi-step process that requires assembly of RNA polymerase (Pol) II and the general transcription factors into the closed complex. Then, the DNA surrounding the TSS is melted and inserted into the Pol II active center cleft to form the open complex (OC), the starting point for RNA synthesis. The OC is hence a central intermediate in the initiation-elongation transition and insights into its structure are crucial for understanding the molecular mechanism of transcription initiation.X-ray crystallography has provided detailed insights into the architecture of Pol II in different functional states, but structural studies of transcription initiation have been hindered by the flexibility of Pol II initiation complexes. As a consequence, the structure of the OC remains unknown.Here, we employ single-molecule Forster Resonance Energy Transfer (smFRET) and Nano-Positioning System (NPS)analysis [1] to determine the 3-dimensional architecture of a minimal OC consisting of Pol II, promoter DNA, TBP, TFIIB, and -IIF. Briefly, by using smFRET, we measure distances within double labeled OCs and the NPS computes 3-dimensional position probability densities of unknown sites on upstream DNA, TATA-DNA, TBP, and TFIIB, which allows us to build a model of the OC.We show that in the OC, TBP and TATA-DNA reside above the Pol II cleft between clamp and protrusion. Downstream DNA is dynamically loaded into the cleft and unloaded from the cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it was located in the closed complex. Hence, our results define the overall structural changes during the initiation-elongation transition, and reveal a key role for the intrinsic flexibility of TFIIB in accommodating these changes.[1] Muschielok et al., Nature Methods, 5, 965 (2008).

Core promoter elements recognized by transcription factor IIB

The general transcription factor TFIIB (transcription factor IIB) plays a critical role in the assembly of the RNA polymerase II pre-initiation complex. TFIIB can make sequence-specific DNA contacts both upstream and downstream of the TATA box. This has led to the definition of two core promoter BREs (TFIIB-recognition elements), one upstream [BRE(u) (upstream BRE)] and one downstream of TATA box [BRE(d) (downstream BRE)]. TFIIB-BRE(u) and TFIIB-BRE(d) contacts are mediated by two independent DNA-recognition motifs within the core domain of TFIIB. Both the BRE(u) and the BRE(d) modulate the transcriptional potency of a promoter. However, the net effect of the BREs on promoter activity is dependent on the specific blend of elements present within a core promoter.

Mapping the Location of TFIIB within the RNA Polymerase II Transcription Preinitiation Complex: A Model for the Structure of the PIC

Biochemical probes positioned on the surface of the general transcription factor TFIIB were used to probe the architecture of the RNA polymerase II (Pol II) transcription preinitiation complex (PIC). In PICs, the TFIIB linker and core domains are positioned over the central cleft and wall of Pol II. This positioning is not observed in the smaller Pol II-TFIIB complex. These results lead to a new model for the structure of the PIC, which agrees with most previously documented protein-DNA interactions within Pol II and archaea PICs. Specific interaction of the TFIIB core domain with Pol II positions and orients the promoter DNA over the Pol II central cleft, and TBP-DNA bending leads to bending of the promoter around the surface of Pol II. The TFIIF subunit Tfg1 was found in close proximity to the TFIIB B finger, linker, and core domains, suggesting that these two factors closely cooperate during initiation.

NMR structure of the hypothetical protein NMA1147 from Neisseria meningitidis reveals a distinct 5-helix bundle.

NMR structure of the hypothetical protein NMA1147 from Neisseria meningitidis reveals a distinct 5-helix bundle.

Potential targets for HSF1 within the preinitiation complex

Protein-protein interactions between human heat shock transcription factor 1 (hHSF1) and general transcription factors TFIIA-gamma, TFIIB, TBP, TAF(II)32, and TAF(II)55 and positive coactivator PC4 were characterized in order to identify potential targets of contact in the transcriptional preinitiation complex. These contacts represent one of the final steps in the signal transfer of heat stress to the transcriptional apparatus. TATA-binding protein (TBP) and transcription factor IIB (TFIIB) were identified as major targets for HSF1 transcriptional activation domains AD1 and AD2 based on in vitro interaction assays. TBP showed affinity for AD2 and a fragment containing AD1, while the core domain of TFIIB interacted primarily with the AD1 fragment. Interactions were also detected between full-length HSF1 and the small subunit (gamma) of TFIIA. PC4 interacted weakly with HSF2 and showed even less affinity for HSF1. Coimmunoprecipitation of transiently expressed TBP in HeLa cells demonstrated that HSF1 AD2 and AD1+AD2 are able to bind TBP in vivo. Assays based on transcriptional interference confirmed predictions that both TBP and TFIIB can interact with HSF1 activation domains in HeLa cells. The negative regulatory region (NR) of HSF1 did not interact with any general factors tested in vitro but did bind TFIID in nuclear extracts through contacts that probably involve TATA associated proteins (TAFs). These results suggest a model for transcriptional regulation by HSF1 that involves a shift between formation of dysfunctional TFIID complexes with the NR and transcriptionally competent complexes with the C-terminal activation domains.

Human general transcription factor TFIIB: conformational variability and interaction with VP16 activation domain.

Human TFIIB, an essential factor in transcription of protein-coding genes by RNA polymerase II, consists of an amino-terminal zinc binding domain (TFIIBn) connected by a linker of about 60 residues to a carboxy-terminal core domain (TFIIBc). The TFIIB core domain has two internally repeated motifs, each comprising five alpha-helices arranged as in the cyclin box. Compared to the crystal structure of TFIIBc in complex with TBP and a TATA-containing oligonucleotide, the NMR-derived solution structure of free TFIIBc is more compact, with a different repeat-repeat orientation and a significantly shorter first helix in the second repeat. Analysis of backbone 15N relaxation parameters indicates the presence of relatively large amplitude, nanosecond time-scale motions in the TFIIBc interrepeat linker and structural fluctuations throughout the backbone. Interaction of TFIIBc with the acidic activation domain of VP16 or with TFIIBn induces 1H-15N chemical shift and line width changes concentrated in the first repeat, interrepeat linker and the first helix of the second repeat. These results suggest that TFIIB is somewhat pliable and that the conformation of the C-terminal core domain can be modulated by interaction with the N-terminal zinc binding domain. Furthermore, binding of the VP16 activation domain may promote TFIIBc conformations primed for binding to a TBP-DNA complex.

The button test: a small scale method using microdialysis cells for assessing protein solubility at concentrations suitable for NMR.

A simple method has been developed for screening solution conditions to determine conditions under which a protein is soluble at the high concentrations typically used for NMR spectroscopy. The method employs microdialysis cells or 'buttons'. The low sample volume (5 microliters) required for each microdialysis button permits testing of a wide range of solution conditions and temperatures with high protein concentrations, using a small amount of protein. Following precipitation of several NMR samples of the C-terminal core domain of human TFIIB, the microdialysis button screen facilitated identification of conditions in which precipitation of the TFIIB core domain was eliminated. The microdialysis button method for screening solution conditions is generally applicable and has been used to permit rapid identification of suitable NMR sample solution conditions for proteins involved in transcription and cell adhesion.

Solution structure of the c-terminal core domain of human TFIIB: Similarity to cyclin A and interaction with TATA-binding protein

TFIIB is an essential component of the machinery that transcribes protein-coding genes. The three-dimensional structure of the human TFIIB core domain (TFIIB c) has been determined using multidimensional heteronuclear magnetic resonance spectroscopy. The molecule consists of two direct repeats that adopt similar α-helical folds, conferring pseudo-twofold symmetry. An extensive, central basic surface including an amphipathic a helix is critical to the function of TFIIB as a bridge between the TBP-promoter complex and RNA polymerase II and associated general and regulatory transcription factors. Similarities between the TFIIB c and cyclin A folds indicate that elements of the eukaryotic cell cycle control apparatus evolved from more fundamental transcriptional control components, demonstrating a link between the transcription and cell cycle molecular machineries.