Recombinant TATA-box-binding protein (tbp-1)

What is TATA-box-binding protein (TBP) and what is its role in transcription?

TBP is a basal transcription factor that plays an essential role in transcription initiation in both eukaryotes and archaea. It functions as a critical component of the RNA polymerase II transcription machinery, initiating gene transcription by recognizing and binding to the TATA box in promoter regions. This binding event represents one of the first steps in gene transcription, as it recruits other transcription factors and eventually RNA polymerase II to transcribe DNA into mRNA .

TBP's fundamental role involves creating a nucleation point for the assembly of the pre-initiation complex through specific DNA recognition and subsequent protein-protein interactions with other transcriptional components.

How does TBP differ from TBP-related factors (TRFs)?

While TBP was initially thought to be the only protein of its kind, research has identified TBP-related factors (TRFs) such as TRF1 and TRF2 in metazoans. These factors share structural similarities with TBP but appear to regulate different subsets of genes .

This diversity appears to represent an evolutionary adaptation that accommodates the increased complexity of gene expression patterns in higher organisms.

What experimental properties characterize properly folded recombinant TBP?

Properly folded recombinant TBP should exhibit specific structural and functional characteristics that mirror the native protein. Key properties include:

• DNA binding ability: Correctly folded TBP should specifically bind TATA box-containing DNA sequences .

• Secondary structure composition: The protein should display characteristic secondary structure elements that can be verified through spectroscopic methods like circular dichroism (CD) .

• Protein-protein interactions: Functional recombinant TBP should interact with known binding partners such as TFIIA, TFIIB, and other transcription factors .

• Salt stability: Studies of halobacterial TBP indicate that properly folded TBP remains stable across a wide range of salt concentrations .

For example, histidine-tagged halobacterial TBP produced in E. coli initially adopts a denatured conformation but can be refolded to a native state. CD spectroscopy confirms this refolding, and the properly folded protein demonstrates binding to TATA box-containing DNA fragments .

How does TBP induce structural changes in intrinsically disordered proteins?

TBP can induce significant structural changes in intrinsically disordered protein regions through specific protein-protein interactions. A notable example is its interaction with the activation function 1 (AF1) domain of the glucocorticoid receptor (GR), which normally exists as an unstructured collection of random coil configurations when expressed independently .

When TBP interacts with GR AF1, it induces the acquisition of significant helical content, monitored through Fourier transform infrared and NMR spectroscopies, and by proteolytic digestion experiments. This structural transformation primarily occurs at the expense of random coil conformation, supporting an induced-fit mechanism model .

This ability to induce structure in otherwise disordered domains represents an important regulatory mechanism in transcription factor function, potentially allowing for context-dependent activation through conformational changes.

What is known about the dynamics of TBP binding to the TATA box?

TBP binding to the TATA box exhibits complex dynamics with multiple interesting features:

• Binding orientation: TBP can bind to the TATA box in two different orientations. For the adenovirus major late (AdML) promoter, approximately 67% of TBP binds in the correct orientation and 33% in the opposite orientation .

• Influence of cofactors: TFIIA improves the alignment of TBP on the promoter site, increasing the percentage of properly oriented TBP molecules to 84% .

• Time-dependent changes: Longer incubation times lead to improved TBP orientation on the TATA box .

• Multiple FRET states: Single-pair Förster Resonance Energy Transfer (spFRET) measurements reveal abrupt transitions between multiple FRET states, indicating conformational changes during the binding process .

These dynamics suggest that TBP-DNA interactions are not simple one-step binding events but involve multiple conformational states and potential rearrangements influenced by both time and the presence of additional factors.

How do mutations affect TBP's interactions with other transcription factors?

Comprehensive mutational analysis has identified specific TBP surfaces critical for interactions with various transcription factors:

• BTAF1 interaction: Two major regions of TBP are important for BTAF1 binding. The first maps to the upper side of the first TBP repeat in the area of helix 2, with charged residues R235, K236, R239, and K243 being particularly sensitive to mutation. The second region includes residues in the loop between helix 2 and strand 1' and in strand 1' itself (L244, K249, F250, and F253) .

• Differential effects: TBP mutations can selectively affect interactions with different partners. For example, some mutations specifically disrupt BTAF1 binding while preserving interactions with other factors like TFIIA or NC2 .

• Functional consequences: Mutations in the convex surface of TBP (helix 2 region) that disrupt BTAF1 binding in solution also impair formation of BTAF1-TBP-DNA complexes. In contrast, mutations in the concave DNA-binding surface often still allow BTAF1-TBP-DNA complex formation despite affecting direct DNA binding .

This mutational mapping provides valuable tools for dissecting separate functions of distinct TBP complexes both in vitro and in vivo.

What are the optimal methods for producing functional recombinant TBP?

Production of functional recombinant TBP typically involves several key steps:

The protocol must be optimized for each specific TBP variant, as proteins from different species (e.g., archaeal vs. eukaryotic TBP) may have distinct folding requirements.

How can researchers detect and analyze TBP-DNA complexes?

Several complementary techniques are employed to detect and characterize TBP-DNA interactions:

• Electrophoretic mobility shift assays (EMSA): This technique identifies TBP-DNA complexes as slower-migrating bands compared to free DNA. It can be extended to detect higher-order complexes, such as BTAF1-TBP-DNA ternary complexes .

• Single-pair FRET (spFRET): This approach uses fluorescently labeled DNA probes to monitor TBP binding at the single-molecule level. It can distinguish between different conformational states and binding orientations, providing insights into binding dynamics not accessible through bulk measurements .

• Specificity controls: The specificity of complexes can be confirmed through supershift experiments with antibodies against TBP or its binding partners .

• ATP sensitivity assays: For complexes involving ATP-dependent factors like BTAF1, ATP sensitivity can be used to validate complex identity .

These methods can be combined to provide comprehensive characterization of TBP-DNA interactions under various experimental conditions.

What approaches help distinguish TBP's direct versus indirect effects on transcription?

Distinguishing direct versus indirect effects of TBP on transcription requires multiple complementary approaches:

• Mutational analysis: Comprehensive testing of surface-exposed TBP residues can identify specific interfaces required for different interactions. For instance, analysis of 85 TBP mutants with 57 mutated surface residues has mapped the surfaces contacted by BTAF1 .

• Comparative binding assays: Testing a panel of TBP mutants for interactions with different partners (e.g., BTAF1, TFIIA, NC2) can identify residues with differential effects, helping to isolate specific interaction surfaces .

• Ternary complex formation: Assessing the ability of TBP mutants to form complexes with both DNA and cofactors can reveal unexpected functional relationships. For example, some TBP mutants defective in BTAF1 binding in solution can still form BTAF1-TBP-DNA ternary complexes .

• DNA binding specificity assays: Testing TBP binding to both TATA and non-TATA sequences in the presence of different cofactors can illuminate how these factors modify TBP's intrinsic specificity .

These approaches collectively help delineate TBP's direct contributions to transcriptional regulation.

How does BTAF1 modify TBP's DNA-binding properties?

BTAF1 dramatically alters TBP's DNA-binding properties in several remarkable ways:

• Rescue of DNA-binding defects: BTAF1 can enable DNA binding for the majority of TBP mutants with defects in their DNA-contacting surface. Only mutations in critical residues (F288K, K309E, R203A) prevent this rescue effect .

• TATA-box independence: While wild-type TBP cannot bind to non-TATA DNA (e.g., TATAAAAG mutated to CGCAAACG), addition of BTAF1 fully rescues this interaction. This dramatically changes TBP's sequence specificity requirements .

• Surface engagement: BTAF1 appears to engage exclusively the upper convex surface of TBP when bound to DNA, rather than the concave DNA-binding surface .

• Dual functionality: Despite its ability to stabilize TBP-DNA interactions, BTAF1 can also dissociate these complexes through its ATPase function, suggesting a complex regulatory role .

This BTAF1-mediated plasticity in TBP-DNA interactions may contribute to promoter-specific regulation and transcriptional diversity.

How do different transcription factors compete for binding to overlapping TBP surfaces?

Multiple transcription factors interact with overlapping surfaces on TBP, creating a complex regulatory network:

• Shared binding regions: Factors including BTAF1, TFIIA, NC2, Brf1, and TAF1 interact with overlapping surfaces of TBP, particularly in the region of helix 2 on the convex side .

• Antagonistic relationships: Some factors directly compete for TBP binding. For example, both TFIIA and NC2 have been suggested to antagonize BTAF1 or Mot1p binding to TBP .

• Differential effects on DNA binding: Despite competing for similar TBP surfaces, different factors have distinct effects on TBP-DNA interactions. BTAF1, TFIIA, NC2, and TFIIB all interact with TBP but differ in their ability to stabilize TBP binding to DNA .

• Selective mutants: Specific TBP mutations have been identified that differentially affect binding of BTAF1, TFIIA, and NC2, providing tools to dissect separate functions of distinct TBP complexes both in vitro and in vivo .

This complex interplay suggests that the composition of TBP-containing complexes is dynamically regulated through competitive and cooperative interactions among multiple factors.

What is the evolutionary significance of TBP-related factors in metazoans?

The discovery of TBP-related factors (TRFs) in metazoans has significant evolutionary implications:

These findings suggest that metazoans evolved multiple TBP variants to accommodate the vast increase in genes and expression patterns during evolution, contributing to the complex regulatory networks required in multicellular organisms.

How can researchers interpret contradictory findings in TBP-DNA binding studies?

Researchers often encounter seemingly contradictory results regarding TBP-DNA interactions. These can be reconciled through several analytical approaches:

• Context-dependent effects: TBP's DNA binding properties are highly influenced by its interaction partners. For example, wild-type TBP cannot bind non-TATA DNA, but BTAF1 enables this interaction .

• Orientation analysis: TBP can bind the TATA box in two orientations. Single-molecule techniques reveal that approximately 67% of TBP binds the AdML promoter in the correct orientation, with TFIIA increasing this to 84%. Failure to account for these multiple binding modes can lead to contradictory interpretations .

• Time-dependent changes: TBP-DNA interactions evolve over time, with longer incubation periods improving TBP alignment on promoter sites. Different experimental timeframes might therefore yield different results .

• Comparative mutation effects: Analyzing how mutations affect binary (TBP-partner) versus ternary (TBP-partner-DNA) complex formation can reveal unexpected functional relationships. For instance, some TBP mutants defective in BTAF1 binding in solution can still form BTAF1-TBP-DNA ternary complexes .

By considering these factors, researchers can develop more nuanced interpretations of apparently conflicting data.

What experimental controls are essential when studying recombinant TBP function?

Rigorous experimental controls are crucial when working with recombinant TBP:

• Folding verification: CD spectroscopy should be used to confirm that recombinant TBP has adopted the correct secondary structure before functional assays .

• Binding specificity: DNA binding should be tested with both TATA-containing and non-TATA control sequences to confirm sequence specificity .

• Complex authenticity: The specificity of detected complexes should be verified through:

  • Supershift experiments with specific antibodies

  • Sensitivity to expected effectors (e.g., ATP sensitivity for BTAF1-containing complexes)

  • Competition with unlabeled DNA sequences

• Multiple mutation types: When analyzing TBP surfaces, multiple types of mutations at the same position (alanine substitutions, charge reversals) should be tested to distinguish specific interaction effects from general structural disruption .

• Comparative analysis: Effects of TBP mutations on multiple interaction partners should be compared to identify specific versus general defects .

These controls help ensure that observed effects genuinely reflect the biological properties of TBP rather than experimental artifacts.

What are the challenges in quantifying TBP-induced structural changes in partner proteins?

Accurately quantifying the structural changes induced by TBP in intrinsically disordered protein regions presents several methodological challenges:

• Baseline characterization: The heterogeneous conformational ensemble of intrinsically disordered regions makes it difficult to establish a clear baseline for measuring changes .

• Spectroscopic interpretation: Techniques like Fourier transform infrared spectroscopy can detect increases in helical content, but precise quantification of different secondary structure elements requires careful analysis and complementary methods .

• Solution conditions: The structural properties of both TBP and its disordered binding partners can be sensitive to solution conditions, requiring careful optimization and standardization .

• Complex formation verification: Ensuring that observed structural changes result from specific TBP interactions rather than non-specific effects requires appropriate controls, such as mutant versions of either partner .

• Functional correlation: Relating observed structural changes to functional outcomes requires additional assays to test whether the induced structure affects transcriptional activity .

These challenges can be addressed through complementary approaches, such as combining spectroscopic techniques with proteolytic digestion experiments and functional assays .