Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways

Abstract

During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein (TBP). Subsequent association of transcription factor B (TFB) with the TBP-DNA complex is followed by the recruitment of the ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly different conditions. Employing single-pair fluorescence resonance energy transfer, we monitored DNA bending by eukaryotic and archaeal TBPs in the absence and presence of TFB in real-time. We observed that the lifetime of the TBP-DNA interaction differs significantly between the archaeal and eukaryotic system. We show that the eukaryotic DNA-TBP interaction is characterized by a linear, stepwise bending mechanism with an intermediate state distinguished by a distinct bending angle. TF(II)B specifically stabilizes the fully bent TBP-promoter DNA complex and we identify this step as a regulatory checkpoint. In contrast, the archaeal TBP-DNA interaction is extremely dynamic and TBP from the archaeal organism Sulfolobus acidocaldarius strictly requires TFB for DNA bending. Thus, we demonstrate that transcription initiation follows diverse pathways on the way to the formation of the pre-initiation complex.

Figure 1.

TBP-induced bending of the promoter DNA and DNAs used for the FRET-based single-molecule assay. (A) Structural alignment of TBP from Methanocaldococcus jannaschii (dark blue), Sulfolobus acidocaldarius (light blue) and Saccharomyces cerevisiae (red). (B) Ternary complex of ScTBP (orange), ScTF(II)B (light green) and DNA. Two sets of two conserved phenylalanines (cyan) are inserted in the minor groove of the DNA backbone (PDB:1C9B). (C) Principle of the FRET based bending assay. The TATA-box is framed by a donor (Cy3b or ATTO532) and an acceptor dye (ATTO647n) resulting in a low FRET signal. Once a TBP protein bends the DNA, the distance between the fluorophores is reduced and consequently the FRET efficiency increases. The change in FRET can be analysed on the single-molecule level. (D) Promoter sequences used in this study. The upper non-template strand is biotinylated at the 5′ end and internally labelled with Cy3b or ATTO532. The bottom template strand is labelled with the acceptor dye ATTO647n internally or at the 5′ end. The cyan box denotes the TATA-box and the grey box the BRE element. The bending studies for the archaeal model systems were carried out either with the Sulfolobus spindle-shaped virus 1 (SSV) T6 gene promoter (DNA_SSV) or a mutated version of this (DNA_SSV-BRE). This DNA contains a mutated BRE element that prevents binding of TFB. Experiments using ScTBP and ScTF(II)B involved the eukaryotic H2B-J core promoter (DNA_H2B).

Figure 2.

Transcription initiation factors induce promoter DNA bending. (A) Donor–acceptor labelled promoter DNA exists in a uniform state with mean FRET efficiencies of 0.25 ± 0.07 and 0.27 ± 0.07 for the DNA_SSV in N70 buffer and DNA_SSV in N65 buffer, respectively. The eukaryotic promoter DNA_H2B shows a major population with a mean efficiency of 0.35 ± 0.06 and a minor subpopulation centered at 0.20 ± 0.05. The minor population remains constant even after addition of TFs and is an inherent property of the labelled DNA construct. The data sets were fitted with a Gaussian fit and the standard deviations are given. (B) Addition of MjTBP (1 μM) to the respective DNA resulted in an additional population with a mean FRET efficiency of 0.49 ± 0.09 (shaded area) corresponding to a bent fraction of the promoter while the FRET efficiency of the unbent DNA fraction remains constant at 0.25 ± 0.08. In contrast, addition of SaTBP (6 μM) to the promoter DNA did not cause a shift of the population towards higher FRET efficiencies (0.28 ± 0.07). Incubation of the promoter DNA_H2B with ScTBP (20 nM) broadens the FRET distribution towards higher values indicating TBP-induced DNA bending (0.52 ± 0.17). (C) While the addition of MjTFB (2 μM) does not alter the FRET distribution significantly (E_{low FRET} = 0.27 ± 0.09 and E_{high FRET} = 0.48 ± 0.09), the addition of SaTFB (5 μM) facilitates bending of the promoter DNA and an almost complete shift to a high FRET population occurs (E_{low FRET} = 0.27 ± 0.09 and E_{high FRET} = 0.49 ± 0.11). The presence of ScTF(II)B gives rise to a second high FRET population (E_{intermediate FRET} = 0.51 ± 0.16 and E_{high FRET} = 0.68 ± 0.08).

Figure 3.

Influence of physiological parameters on TBP-induced bending of promoter DNA. The influence of pH, salt concentration and temperature on the formation of the bent DNA–TBP complex was investigated by comparing the fraction of low to high FRET populations. Measurements involving SaTBP (5 μM) were carried out in the presence of 5 μM SaTFB as this is mandatory for bending. pH and salt dependency studies were performed using confocal solution measurements at 35°C (archaeal proteins) or room temperature (Saccharomyces cerevisiae) while the temperature dependence was investigated on immobilized molecules via TIRF microscopy. (A) pH dependency: DNA bending is marginally affected by variation of the pH value for Methanocaldococcus jannaschii (1 μM MjTBP), Sulfolobus acidocaldarius (5 μM SaTBP, 5 μM TFB) and S. cerevisiae (30 nM ScTBP). (B) Salt dependency: an increase in salt concentration (potassium acetate, KAc) results in a higher fraction of bent DNA for the archaeal systems (1 μM MjTBP, 5 μM SaTBP and 5 μM SaTFB) suggesting a mainly hydrophobic interaction between MjTBP and the DNA. ScTBP-induced DNA bending (30 nM ScTBP in T72) in the eukaryotic system is diminished at higher salt concentrations indicating that the protein–DNA contact is based on electrostatic interactions. (C) Temperature-dependency: MjTBP as well as SaTBP/SaTFB (for technical reasons, measurements above 50°C for S. acidocaldarius could not be performed) show increased bending efficiency with higher temperature in agreement with their thermophilic growth conditions. The formation of the bent DNA population in the S. cerevisiae system is strongly reduced at higher temperatures. The lines in all graphs are only present as a guide to the eye.

Figure 4.

Temperature-dependent dynamics of the archaeal TBP–promoter DNA interaction. Exemplary transients of immobilized promoter DNA molecules upon DNA binding and dissociation and analysis of the kinetics. (A) Exemplary traces of DNA_SSV in the presence of 5 nM MjTBP at 22 and 60°C. Rapid fluctuations between the low and high FRET states are observed suggesting bending and unbending of the DNA upon rapid association and dissociation of MjTBP. (B) Dwell time histograms from a Hidden-Markov-Model analysis of the FRET traces for the respective conditions. The results were fitted with a mono-exponential decay (for details see Supplementary Information). The dwell time and by inference the complex lifetime of MjTBP is almost independent of the temperature (τ_22°C = 0.18 ± 0.01 s and τ_60°C = 0.14 ± 0.01 s) while the off-time decreases with increased temperature (τ_22°C = 19.2 ± 1.2 s and τ_60°C = 1.14 ± 0.04 s, both with 5 nM MjTBP). (C) Exemplary traces of DNA_SSV in the presence of 6 μM SaTBP at 22 and 50°C. Similar dynamics can be observed in the presence of SaTBP (6 μM)/SaTFB (2.1 μM). (D) The dwell time analysis of transients recorded using SaTBP (in the presence of 2.1 μM SaTFB) yielded comparable results. The dwell time in the bent state remains constant at higher temperature (τ_22°C = 2.1 ± 0.1 s and τ_60°C = 2.5 ±0.8 s) while the delay between two bending events is dramatically shortened with higher temperature (τ_22°C = 167 ± 15 s and τ_60°C = 20.3 ± 0.6 s). The dynamics immediately stop once the measurement chamber is flushed with fresh buffer indicating that the initiation factors of Sulfolobus acidocaldarius and Methanocaldococcus jannaschii are not stably bound but that the bending events are caused by different TBP molecules and not by one molecule inducing different bending states.

Figure 5.

Dynamics of promoter DNA bending in the presence of TBP and TF(II)B from Saccharomyces cerevisiae. (A) FRET time traces of the promoter DNA only (top), promoter DNA after incubation for 20 min at room temperature with 20 nM ScTBP (middle) and after additional incubation with ScTF(II)B (500 nM). ScTBP remains associated with the promoter DNA for minutes. The traces show different interconverting FRET states exhibiting at least two different bent states, which we assign to the intermediate and fully bent state (E_unbent = 0.32, E_intermediate = 0.60 and E_{fully bent} = 0.75). (B) Dwell time analysis of the different DNA conformations in the presence of ScTBP and ScTBP/ScTF(II)B. Addition of ScTF(II)B leads to a slight increase of the dwell time of the intermediate state (from 0.31 ± 0.02 to 0.42 ± 0.03 s) and more significant increase of the dwell time of the fully bent state (from 0.44 ± 0.04 to 0.83 ± 0.01 s). The lifetime of the unbent state is reduced from 0.24 ± 0.02 to 0.18 ± 0.01 s. See Supplementary Figure S10 for the raw histograms, dwell-time histograms and the mono-exponential fits. (C) Relative transition frequency between the three different states shows that the transition from the unbent to the fully bent state and the reverse transition are extremely rare and these transitions are considered as artifacts arising from the limited temporal resolution of 50 ms. This suggests that the three states are connected linearly as shown in the model presented in panels (D) and (E). (D) Linear model of the ScTBP action in the absence (D) and presence of ScTF(II)B (E). The enhanced lifetime of the fully bent state in the presence of ScTF(II)B is accompanied by a decrease of the transition rate (clearly changed rates highlighted in green) from the fully bent to the intermediate bent state (see Supplementary Figure S10 for details of the transition rate calculation).

Figure 6.

ScTF(II)B does not change the overall architecture of the binary ScTBP–promoter DNA complex. (A) In order to measure a FRET signal directly informing about the association of ScTBP with the promoter DNA, a ScTBP mutant was labelled with the donor dye ATTO532 and the acceptor dye ATTO647n was attached to the oligonucleotide through a dT base (position highlighted in red) to the H2B promoter DNA, which can be immobilized via the biotin modification. (B) Typical fluorescent transient acquired from the ScTBP–DNA–FRET pair. The fluorescence and hence the FRET efficiency remains constant until the acceptor bleaches (11 s) followed by the bleaching of the donor (16 s). (C) FRET efficiency histograms show that the presence of ScTF(II)B (500 nM) does not have any significant influence on the main FRET efficiency (−TF(II)B: E = 0.27 ± 0.01, +TF(II)B: E = 0.26 ± 0.01 (mean ± SE) ). (D) Confocal population analysis reveals a lifetime of the complex of 12.2 ± 1.1 min (see Supplementary methods).

Figure 7.

TBP recruitment and DNA bending is a regulatory checkpoint during transcription initiation in archaea and eukaryotic transcription systems. In archaea, the promoter DNA is uniformly bent while eukaryotic TBP induces multiple bent states. However, depending on the archaeal organism, TBP is sufficient (Methanocaldococcus jannaschii) or both general TFs, TBP and TFB, are required for promoter DNA bending (Sulfolobus acidocaldarius). Transcriptional regulation can be achieved by either preventing or enhancing TBP association with the DNA. Transcriptional activators (indicated with a red plus sign) like Lrs14 and Ptr2 enhance binding of archaeal TBP to the promoter DNA. In contrast, negative regulators like Mot1 and NC2 (indicated by a red minus sign) prevent archaeal TBP interaction with the DNA. The general eukaryotic transcription factor TF(II)A stabilizes the eukaryotic TBP–DNA complex as does ScTF(II)B. ScTF(II)B exerts its stabilising and potentially regulatory effect by a shift in binding equilibrium towards the transcriptional active, fully bent DNA state. ScTF(II)B acts in a sequential manner to eukaryotic TBP in the transcriptional system of RNAPII while DNA bending requires co-action of TBP and TFB (called Brf in the RNAPIII system) in the RNAPIII system and S. acidocaldarius. TBP and TFB are inherently co-acting in the transcriptional apparatus of the eukaryotic RNAPIII system as they are part of the multidomain complex TF(III)B.