HUMEDALES RECONOCIDOS POR EL DISTRITO

above shifts the focus of NPS from determining fluorophore positions to the structurally more relevant positions and orientations of macromolecules relative to each other. In the next section, this framework will be applied to determine the gross structure of the initial transcribing complex of Pol II.

6.6 Application III - Position and orientation of TBP in the initial

transcribing complex

This section deals with the coarse positioning of the TATA binding protein (TBP) within the initial transcribing complex (ITC), which is important for the detailed understanding of the initiation phase of eukaryotic transcription.

6.6.1 NPS inference

The analysis is based on experimental data measured by Barbara Treutlein, Joanna An- drecka and Monika Holzner. The measurements were carried out with stalled Pol II initial transcribing complexes (section 5.1.1). The analysis should be regarded as preliminary, as the ITC project is still subject of current research.

FRET Network structure and prior assumptions

The satellite fluorophores were attached on the template DNA and the Rpb4/7 heterodimer to the positions tDNA(-10), tDNA(+3), tDNA(+7), tDNA(+12), and Rpb7(C150). The antennas were placed on the nontemplate DNA at both ends of the TATA box at positions ntDNA(-30) and ntDNA(-37), as well as on TBP at the position TBP(S159C). It was assumed that these labeling sites are located on a rigid macro- molecule, the TBP/TATA subcomplex, consisting of TBP and the bent DNA double strand in the region of the TATA box. An additional antenna fluorophore was placed at position ntDNA(-20) on the nontemplate DNA strand, which was assumed to be base paired with the template DNA and thus part of a locally rigid upstream DNA double strand segment.

The TBP/TATA subcomplex and the upstream DNA segment were docked to Pol II by means of 20 FRET efficiency measurements in total, which were acquired with various fluorophores (Alexa 647, TMR and Cy3) (figure6.38). The data is listed in theappendix

Figure 6.38:Initial tran-

scribing complex FRET network. 20 FRET efficiencies (dotted lines) were measured between fluorophores attached to Pol II, the TBP/TATA subcomplex and the upstream DNA. Fluo- rophores attached to the same labeling site were treated as completely independent, i.e. were described by separate positions and average transition dipole moment orientations.

Figure 6.39: Satellite position priors. The volumes accessible to the satellite fluorophores at- tached to Pol II are shown in the top (left) and side view (right). The fluorophore position priors were flat within these volumes.

II in tables 3(fluorescence anisotropies), 6(FRET efficiencies) and 7(isotropic Förster distances).

The volumes accessible to the fluorophores were computed based on the Pol II elon- gation complex structure (Kettenberger et al.,2004, PDB-ID: 1Y1W) (figure 6.39), the structure of TBP bound to DNA with the TATA sequence (Nikolov et al.,1995, PDB-ID: 1VOL) (figure6.40a), and a structure of double-strand DNA in B-form (Drew et al.,1981, PDB-ID: 1BNA) (figure 6.40b). It was hence assumed that the structure of the Pol II enzyme is identical in the elongation and initial transcribing complexes. This applies in particular to the position of the Rpb4/7 heterodimer and the template DNA strand. Fur- ther, the local structure of the DNA bent by TBP at the TATA sequence was assumed to be unchanged by possible interactions with the other ITC components. Also the local structure of the upstream DNA was assumed to be double stranded and not bent within two lengths of the carbon chain linker used for attachment of the fluorophore at the po- sition ntDNA(-20) (figure 6.40b). In this way, it was possible to compute the marginal position density of the attachment atom of ntDNA(-20).

In contrast to the calculations in the sections6.2and6.4, each fluorophore was described by a separate set of parameters even when different fluorophores were attached to the same labeling site. Completely uninformative priors were used in the average transition dipole moment parameters of the fluorophores, and the fluorophore position priors were flat within the accessible volumes.

The docked macromolecule parts were described by the positions and orientations of reference frames (figure6.40). The reference frame position priors were flat within large boxes, which contained the region that was accessible to the upstream DNA and the TBP/TATA subcomplex5_{, while the orientation priors were completely uninformative.}

6.6 Application III – Position and orientation of TBP in the ITC

Figure 6.40: ITC, antenna position priors. The accessible volumes of the antenna fluorophores

are shown for the TBP/TATA subcomplex (a) and the upstream DNA segment (b). The fluorophore position priors were flat within these volumes. The frames of reference, (x₍k), y(k), z(k)) (k∈ {1,2}), attached to the rigid macromolecule parts are shown as well.

The accessible volume of TBP(S159C) was computed by using an unusually long linker, since the fluorophore is attached to the end of an amino acid sequence lacking secondary structure in the crystal. It was hence assumed that this sequence might be disordered and therefore at a different position in reality, which elongated the linker effectively.

Inference results and preliminary modeling

The inference of th 77 model parameters was performed with nested sampling using 1000 objects. The position of the TBP/TATA subcomplex was visualized by displaying the marginal position posterior density of the Cαcarbon atom of isoleucin 168 (I168) in the TBP structure, which is located at the interface of TBP and DNA in close proximity to the bent DNA. To estimate the position of the upstream DNA at register -20, the marginal position posterior density of the attachment site ntDNA(-20) was computed (figure6.41a).

To find the approximate orientation of the TBP/TATA subcomplex, the marginal posi- tion posterior densities of three points located far from I168 were computed. Two points were situated approximately on the axes defined by the DNA double strands upstream and downstream of the TATA box sequence, while the third point was placed on the opposite side of TBP, above the bent DNA. Since the marginal densities of these points were very large and hence the orientation uncertainty was high, the densities were used only as a guide to orient the TBP/TATA subcomplex (figure6.41b). Finally, an existing biochemical model of the minimal pre-initiation complex (Kostrewa et al.,2009) was used as a rough guideline for the orientation of the DNA in between the TATA box and Pol II. The orientation of the DNA segment between the transcription bubble and the TATA box was not evaluated, since it contained only one attachment site.

Inconsistent FRET efficiency data was not observed, as confirmed by comparison of each measurement’s marginal likelihood factors and the distribution of the respective expected FRET efficiencies (not shown).

6.6.2 Discussion

The position of the TBP/TATA subcomplex was localized by FRET efficiency measure- ments above the cleft of Pol II with an accuracy of 9, 17 and 18 Å (standard deviation probability density maximum, which could not be reached by the TBP/TATA subcomplex and the upstream DNA at register -20 when the complexes were assembled correctly.

Figure 6.41: A preliminary ITC model. Stereo images of the preliminary ITC model. In (a), the approximate position of the TBP/TATA subcomplex (TBP: magenta ribbons, template DNA: blue, nontemplate DNA: cyan cartoons) located above the Pol II (1Y1W PDB struc- ture) is shown. The model was built by considering the density of I168 (68% credible volume, yellow) shown together with the density of ntDNA(-20) (68% credible volume, red). Both positions are shown as spheres in the structure and marked by arrows. The position of the ntDNA(-20) density indicates that the DNA strand between TBP and Pol II must be bent, as it is expected from the approximate exit site of the upstream template DNA (blue cartoon inside of Pol II). In (b), a detailed view of the 68% credible volumes of of the I168 position and three other points in the TBP/TATA reference frame is shown. Each point is displayed as a sphere in the respective color of the credible volume surface and marked by an arrow. The TBP/TATA subcomplex is shown as magenta ribbons (TBP, barely visible) and blue/cyan cartoons. The front part of the surfaces are not shown.

6.6 Application III – Position and orientation of TBP in the ITC

Figure 6.42: Pol II center of mass position in the TBP/TATA reference frame. The 68%

credible volume of the marginal position posterior density of the Pol II center of mass ((x₍₀₎, y₍₀₎, z₍₀₎) = (107,52,−2) Å in the coordinate system of 1Y1W) is shown (blue sur- face). The density is banana-shaped since the fluorophores attached to TBP and the DNA (accessible volumes are shown) constitute only a small trilateration basis in that direction. Measurements to additional labels at the positions TBP(A187) or TBP(L337) (arrows) would improve the localization accuracy of the Pol II center of mass, and therefore also the orien- tation accuracy of the TBP/TATA subcomplex relative to Pol II. The direction along the DNA towards the transcription bubble is shown by an arrow.

of the I168 density in the principal directions). The marginal position density of the upstream DNA segment labeled at the site ntDNA(-20) indicates that the DNA between TBP and Pol II is bent towards Pol II. Moreover, a straight DNA double strand between TBP and Pol II would clearly disagree with the inferred orientation of the TBP/TATA subcomplex (figure6.40b, blue density), given the angle of DNA bent by TBP is the same in the ITC and the crystal structure 1VOL.

The DNA could be melted in addition, which is expected since the template DNA strand must reach its exit site separated from the nontemplate DNA in order to form the transcription bubble. The simultaneous melting and bending during transcription initiation, also calledscrunching, was originally proposed in the bacterial RNA polymerase system (Kapanidis et al.,2006; Revyakin et al., 2006). However, the size of the melted region in the ITC is still unclear and subject of current research. This information will be important for both building a correct biochemical model and for the NPS analysis, since it is not known whether the DNA at position ntDNA(-20) is really double stranded as assumed.

Although the position of the TBP/TATA subcomplex was determined well, its orien- tation was less accurate. It would be possible to impove the orientation accuracy by analyzing FRET efficiency measurements between the satellites on the Pol II elongation complex and additional antenna fluorophores on TBP. This becomes evident when one switches to the reference frame of TBP/TATA and displays the marginal posterior posi- tion density of the Pol II center of mass (figure6.42). The density is very elongated, and would shrink if additional labels were attached to the aminoacids A187 or L337 on TBP (sequence given as in 1VOL), which is equivalent to an improved orientation accuracy of TBP/TATA in the Pol II frame of reference.

position densities of points located far from the subcomplex center. It is hence desirable to develop a better method to visualize the orientation of a rigid macromolecule, as discussed already in section6.5.2.

TFIIB might play this role, since it interacts with DNA and TBP (Nikolov et al.,1995), but this should be proven by direct localization of TFIIB in the ITC.

After having shown the preliminary analysis of docking the TBP/TATA subcomplex to PolII, the next section will focus on some general aspects of NPS.