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3.5. Summary and Outlook

The application of smFRET, global docking NPS analysis, and modeling based on x-ray crystallographic information enabled us to determine the molecular architecture of a dy- namic, minimal OC consisting of Pol II, promoter DNA, TBP, TFIIB, and TFIIF. The results uncovered large overall structural changes during the initiation-elongation transi- tion, which are apparently accommodated by the intrinsic flexibility of TFIIB. Moreover, dynamic loading of the downstream DNA into the cleft and unloading from the cleft could be directly observed on a timescale of seconds. The study presents a significant progress towards understanding the mechanism of Pol II transcription initiation, however many open questions remain.

The study presented in this chapter directly suggests further experiments investigating the role of initiation factors in the loading dynamics of melted promoter DNA. The ob- served loading/unloading dynamics should be used as a starting point to explore how transcription factors such as TFIIF and TFIIE stabilize or destabilize the promoter DNA in the cleft, and which structural domains of the protein factors are responsible.

Regarding TFIIF, a first step has been already taken. Endogenous S. cerevisiae TFIIF purified from yeast in complex with Pol II was shown to stabilize the downstream DNA in the Pol II cleft. I verified that recombinant TFIIF is able to reproduce the stabilizing effect on the downstream DNA (Figure 3.5.1). This paves the way for mapping out the regions of TFIIF responsible for the stabilization of promoter DNA, since truncated and mutated constructs of TFIIF can now be tested in a similar way.

Furthermore, the effect of TFIIE and -F on the overall architecture of the OC should be explored and their location within the OC should be determined using smFRET and global NPS analysis.

The nucleic acid scaffold used throughout the experiments presented here consisted of an artificial promoter sequence designed under consideration of the characteristics of a typi- cal mammalian core promoter [246]. However, the sequence of the template DNA strand within the melted region was proposed to have an effect on the architecture and activity of transcription initiation complexes possibly through recognition by TFIIF [71]. There- fore, the architecture of Pol II OCs should be tested using nucleic acid constructs with different, ideally native sequences. Moreover, mammalian and yeast promoters deviate with respect to the distance between TATA box and TSS. Since yeast Pol II and TFs were used in all experiments, the architecture of OCs should be examined using native yeast promoters and compared to the results obtained with the artificial mammalian promoter used here.

Finally, beyond further studies of the Pol II OC, future experiments should use smFRET experiments and global NPS analysis also to explore the architecture of the Pol II closed promoter complex to either verify or correct existing models. Moreover, in the presence of TFIIH, smFRET could be used to follow DNA melting and directly observe the conforma-

Figure 3.5.1.:Recombinant TFIIF fromS. mikatae(tfg1) andS. cerevisiae (tfg2) can exhibit similar stabilizing effect on downstream DNA as endogenous TFIIF. Left: The cartoon il- lustrates the smFRET measurements from satellite T-DNA(+12) to antenna NT-DNA(-23), which was performed in the presence and absence of endogenous and recombinant TFIIF (semi-transparent, gray ellipsoid). An OC is presented schematically and the alternate con- formation of the downstream DNA is sketched with dotted gray lines. Right: FRET efficiency histograms for the measurements in the presence of endogenous (dark green) or recombinant (light green) TFIIF as well as in the absence of TFIIF (blue). Without TFIIF, the majority of OCs contain the downstream DNA in its alternate conformation outside the cleft (high FRET), whereas in the presence of TFIIF (both endogenous and recombinant) the majority of OCs contains downstream DNA inside the cleft (low FRET).

tional changes that occur during the transition from closed to open complex. In this way, the kinetics of this dynamic process could be determined. Since TFIIH is a 10-subunit protein factor, such experiments are complex and challenging. As a promising alternative, the closed to open transition can be studied using the archaeal RNA polymerase initia- tion complex as model system, since it does not require any transcription factor for DNA melting. The archaeal RNA polymerase is both functionally and structurally very similar to eukaryotic Pol II, but has the advantages that it can be prepared as a fully functional recombinant enzyme and that it only requires three transcription factors (TBP, TFB and TFE). As a consequence of the reconstitution from recombinantly expressed subunits, the archaeal RNA polymerase can be site-specifically labeled at any position of the individual subunits and, together with its reduced complexity, it therefore presents a perfect model system for NPS analysis.

Currently, the structural study of Pol II CC using NPS analysis is limited due to a lack of satellite attachment sites within Pol II itself. The position of promoter DNA in the CC is not known, and hence satellites on Pol II are required to localize DNA as well as general TFs. In this study, Rpb4 and 7, which can be recombinantly expressed and reassembled with the Pol II core, were fluorescently labeled via cysteine chemistry. A protocol was recently developed that allows for recombinant expression of Rpb9 and en- dogenous expression of Pol II(∆Rpb9) and their subsequent reconstitution [247]. This protocol should be used in future studies to introduce fluorescent labels into Rpb9 of Pol II. Except for Rpb4, 7 and 9, Pol II cannot be reconstituted from its individual, recombi-

3.5 Summary and Outlook

nantly expressed subunitsin vitro, which impedes labeling via single cysteine mutations. Therefore new labeling strategies will need to be exploited based on the introduction of unnatural amino acids or small peptide tags into specific sites of the Pol II structure, in order to be equipped with a sufficient set of satellites on Pol II.

I believe that smFRET experiments and global NPS analysis applied to the analysis of structure and dynamics of Pol II transcription intermediates will continue to contribute substantially to the progress in our understanding of the mechanisms underlying Pol II transcription.