Recombinant Transcription initiation factor IIB (tfb)

What is Transcription Initiation Factor IIB and what is its role in transcription?

TFIIB is a general transcription factor essential for RNA polymerase II-mediated transcription initiation. It serves as a critical bridge between the TATA-binding protein (TBP) and RNA polymerase II, functioning as part of the preinitiation complex (PIC) assembly. TFIIB associates with TFIID-IIA to form a complex recognized by polymerase II .

Human TFIIB is a single peptide of approximately 33-35 kDa consisting of a zinc ribbon at the N-terminus and a C-terminal core domain composed of α-helices that form two direct repeats . The core domain interacts with TBP at the TATA box and contacts the DNA both upstream and downstream of TATA, while the zinc ribbon recruits RNA polymerase II through interactions with both pol II and TFIIF .

How does TFIIB fit into the RNA polymerase II transcription initiation mechanism?

TFIIB is integral to the step-wise assembly of the preinitiation complex. The process begins with TBP binding to the TATA box, followed by TFIIB association, which creates a platform for subsequent recruitment of RNA polymerase II and TFIIF . The complete assembly sequence is:

• TBP binds to the TATA box

• TFIIB binds to the TBP-DNA complex

• TFIIF-RNA polymerase II complex joins

• TFIIE and TFIIH are recruited

• Transcription initiation occurs

TFIIB stabilizes TBP binding to DNA, as evidenced by DNase I footprint titration studies showing that TFIIB increases TBP's affinity for the TATA box by 2.5-fold under optimal conditions and approximately 10-fold under suboptimal conditions .

What are the structural components of TFIIB and their functions?

TFIIB contains several functional domains with distinct roles in transcription:

The B-finger/B-reader domain is particularly important as it projects into the RNA catalytic center and abuts the initiation site, playing a crucial role in transcription start site selection . The mobility evident from different structural studies suggests this domain functions at several stages, including transcription initiation and clearance of pol II from the promoter .

What is the minimal set of factors required for reconstituting RNAP II promoter binding in vitro?

Research has established that three eukaryotic polypeptides, produced in E. coli and purified to near homogeneity, constitute a minimal set of general transcription factors both necessary and sufficient for specific and stable promoter binding by RNA polymerase II . These polypeptides are:

• Yeast TATA box binding protein (TBP)

• Human general initiation factor TFIIB

• Human RAP30 (small subunit of RAP30/74 or transcription factor IIF)

In this minimalist system, formation of the polymerase-containing complex required only the TATA box of the promoter, not the initiator element (Inr) . This finding provides researchers with a streamlined approach for studying fundamental aspects of transcription initiation.

How can researchers assess TFIIB's role in initial RNA bond formation?

To investigate TFIIB's catalytic role in initial RNA bond formation, researchers have established assays using immobilized preinitiation complexes. One effective methodology involves:

• Isolating immobilized preinitiation complexes

• Directing the condensation of dinucleotide UpA with [α-32P]CTP

• Creating the radioactive RNA product UpApC, which requires formation of a single RNA bond

Control experiments should include:

• Omission of UpA

• Omission of dATP needed for promoter opening

• Addition of α-amanitin (RNA polymerase II inhibitor)

• Excision of the promoter from the template

This assay has revealed that the TFIIB fingertip, particularly a pair of fingertip aspartates that can bind magnesium, acts as a catalytic cofactor for initial RNA bond formation .

What methodologies are effective for studying TFIIB assembly at promoters in vivo?

RNA interference (RNAi)-based systems provide powerful approaches for studying TFIIB assembly at promoters in living cells. An effective methodology includes:

• Ablating endogenous TFIIB expression using RNAi

• Replacing with TFIIB derivatives containing silent mutations that make them refractory to the RNAi

• Analyzing transcriptionally defective TFIIB amino-terminal mutants to observe their effects based on competition with wild-type TFIIB in vivo

• Using chromatin immunoprecipitation to assess promoter occupancy by TFIIB variants

This approach has demonstrated that promoter occupancy by TFIIB is dependent on the association with RNA polymerase II, supporting a model of preinitiation complex assembly in which TFIIB/RNA polymerase II recruitment to the promoter occurs in vivo as a coordinated process .

How does phosphorylation regulate TFIIB function in transcription?

Phosphorylation of TFIIB plays a critical regulatory role in transcription initiation. Research has demonstrated that human TFIIB is phosphorylated at Serine-65 within the B-finger/B-reader domain, and this modification is required for productive transcription initiation .

Key experimental findings include:

• Mutation of TFIIB Serine-65 to Alanine leads to failure in productive transcription at several genes

• This mutation causes accumulation of RNA polymerase II phosphorylated at serine-5 at the gene 5' end

• TFIIB phosphorylation in vivo is significantly inhibited by the CDK inhibitor DRB (5,6-dichloro-1-h-ribofuranosyl-benzimidazole)

These findings confirm the importance of the TFIIB B-finger/B-reader loop in regulating transcription initiation post-PIC assembly and suggest that its function is subject to regulation through phosphorylation. This provides researchers with a potential target for manipulating transcription initiation in experimental systems.

What is the role of TFIIB in gene loop formation and its significance?

TFIIB plays a crucial role in gene loop formation, a process where the promoter and terminator regions of a gene physically interact to form a loop structure. Research indicates that:

• TFIIB enables gene looping by interacting with both promoter and terminator regions

• Gene looping and promoter escape (the transition to productive elongation) may be coupled processes regulated by TFIIB phosphorylation

• Activators facilitate the formation of gene loops through direct interaction with TFIIB

• Gene looping is conferred by activator-dependent interactions between transcription initiation and termination complexes

Is TFIIB universally required for all RNA polymerase II-dependent transcription?

Contrary to the traditional view that TFIIB is universally required for RNAP2 transcription initiation, recent research has revealed a more nuanced picture. Based on bioinformatic analysis and TFIIB knockdown experiments in primary and transformed cell lines:

These findings establish a new paradigm for TFIIB functionality in human gene expression, indicating that different sets of promoters may rely on distinct transcription initiation mechanisms. This has significant implications for understanding gene regulation and developing targeted approaches to manipulate specific transcriptional programs.

How do species-specific differences in TFIIB affect transcription initiation patterns?

Research comparing transcription systems between yeast species has yielded unexpected insights about TFIIB's role in determining transcription start site (TSS) patterns:

• TATA-containing promoters in human cells usually contain a single TSS located ~30 bp downstream of the TATA element

• In contrast, transcription in Schizosaccharomyces pombe and Saccharomyces cerevisiae typically initiates at multiple sites within windows ranging from 30-70 bp or 40-200 bp downstream of TATA elements, respectively

• Contrary to initial hypotheses, genetic and biochemical assays of TFIIB chimeras revealed that TFIIB and the proposed B-finger/reader domain do not play a role in determining these distinct initiation patterns

• These patterns are solely due to differences in RNA polymerase II between species

These findings challenge previous assumptions about TFIIB's role in start site selection and highlight the importance of considering species-specific differences when extrapolating transcription mechanisms across organisms.

What techniques are most effective for expressing and purifying recombinant TFIIB?

For optimal expression and purification of functional recombinant TFIIB, researchers should consider the following methodology:

• Expression system: E. coli has been successfully used to produce functional human TFIIB . BL21(DE3) or similar strains with T7 RNA polymerase are recommended.

• Purification strategy: A multi-step purification approach is typically required:

  • Initial capture using affinity chromatography (His-tag or GST-tag)

  • Ion exchange chromatography for removing charged contaminants

  • Size exclusion chromatography as a final polishing step

• Quality control: Verify the functionality of purified TFIIB by:

  • Assessing its ability to bind TBP-DNA complexes in electrophoretic mobility shift assays

  • Testing its capacity to support transcription in reconstituted systems

  • Confirming proper folding through circular dichroism spectroscopy

Researchers should be aware that small volumes of recombinant TFIIB may occasionally become entrapped in the seal of product vials during shipment and storage , which may affect experimental outcomes if not properly accounted for.

How can researchers design experiments to study TFIIB's role in promoter escape?

To effectively study TFIIB's role in promoter escape, researchers can employ several complementary approaches:

• Mutational analysis: Target the TFIIB fingertip region, particularly the aspartate residues that bind magnesium and function as catalytic cofactors for initial RNA bond formation .

• In vitro transcription assays: Utilize templates with different promoter structures to assess how TFIIB variants affect the transition from initiation to productive elongation.

• Single-molecule techniques: Apply optical trapping or fluorescence resonance energy transfer (FRET) to monitor real-time changes in TFIIB-RNA polymerase II interactions during promoter escape.

• Phosphorylation studies: Investigate the role of TFIIB Serine-65 phosphorylation in promoter escape using phosphomimetic mutants (S65D/E) or phosphorylation-deficient mutants (S65A) .

Such experiments have revealed that the TFIIB fingertip mediates the timing of TFIIB release associated with appropriate promoter escape, providing insights into the molecular mechanisms controlling this critical transition .

How does TFIIB function in virus transcription and what are the implications?

TFIIB plays a crucial role in viral transcription, particularly for herpes simplex virus-1 (HSV-1):

• TFIIB is essential for HSV-1 gene transcription and replication, even though it is dispensable for many human promoters

• TFIIB is known to interact with viral proteins, including Epstein-Barr virus EBNA2 and HIV-1 Vpr

• Downregulation of TFIIB has potent anti-viral effects

This differential requirement for TFIIB between host and viral gene expression suggests potential therapeutic applications. Targeting TFIIB-dependent mechanisms could selectively inhibit viral replication while minimizing effects on host gene expression. Researchers investigating viral transcription mechanisms should consider TFIIB as a key factor that may distinguish viral from cellular transcription processes.

What are common issues when working with recombinant TFIIB and how can they be addressed?

Researchers working with recombinant TFIIB may encounter several challenges:

• Protein stability issues: TFIIB can be prone to degradation.

  • Solution: Add protease inhibitors during purification and storage

  • Store at -80°C in small aliquots to avoid freeze-thaw cycles

  • Include glycerol (10-20%) in storage buffers

• Suboptimal activity in transcription assays:

  • Verify proper folding of the zinc ribbon domain by checking zinc content

  • Ensure the absence of oxidizing agents that could affect the zinc finger structure

  • Test TFIIB under different salt and pH conditions to optimize activity

• Inconsistent binding to TBP-DNA complexes:

  • Remember that TFIIB increases TBP affinity for TATA box by 2.5-fold under optimal conditions but ~10-fold under suboptimal conditions

  • Adjust reaction conditions accordingly when working with different promoters

  • Consider using TBP mutants with enhanced DNA binding to stabilize the complex

• Reduced activity after shipping/storage:

  • Small volumes may become entrapped in vial seals

  • Brief centrifugation before opening vials can help recover entrapped protein

What controls should be included when studying TFIIB function in transcription systems?

When investigating TFIIB function in transcription systems, researchers should include several key controls:

• TFIIB concentration gradient: Establish the optimal TFIIB concentration for your specific transcription system, as both insufficient and excess TFIIB can affect results.

• TFIIB-depletion controls: When using immunodepletion to study TFIIB requirement:

  • Test multiple promoters, including TFIIB-dependent (e.g., viral) and potentially TFIIB-independent promoters

  • Include RNA polymerase III promoters (e.g., Ad2 VAI) as negative controls

  • Verify depletion efficiency by Western blotting

• Reconstitution controls: When adding recombinant TFIIB back to depleted extracts:

  • Use titration to determine the amount needed for activity restoration

  • Include both wild-type TFIIB and well-characterized mutants

  • Test TFIIB from different species to assess evolutionary conservation

• Promoter architecture controls: Include promoters with and without TATA boxes, as TFIIB-DNA interactions differ between these promoter types.

These controls help ensure that observed effects are specifically due to TFIIB function rather than experimental artifacts or indirect effects.