Podcasts about rna polymerase ii

In this episode of the Epigenetics Podcast, we caught up with Sheila Teves from the University of British Columbia to talk about her work on the inheritance of transcriptional memory by mitotic bookmarking. Early in her research career, Sheila Teves focused on the impact of nucleosomes on torsional stress and gene regulation. She also highlights the development of a genome-wide approach to measure torsional stress and its relationship to nucleosome dynamics and RNA polymerase regulation. The conversation then shifts to her focus on transcriptional memory and mitotic bookmarking during her postdoc in the Tijan lab. She explores the concept of mitotic bookmarking, whereby certain transcription factors remain bound to their target sites during mitosis, facilitating efficient reactivation of transcription after cell division. She discusses her findings on the behavior of transcription factors on mitotic chromosomes, challenging the notion that they are excluded during mitosis. She also discusses the differences in binding behavior between the general transcription factor TBP and other transcription factors. Finally, the effect of formaldehyde fixation on the potential to find transcription factors bound to mitotic chromosomes is discussed.   References Teves, S., Henikoff, S. Transcription-generated torsional stress destabilizes nucleosomes. Nat Struct Mol Biol 21, 88–94 (2014). https://doi.org/10.1038/nsmb.2723 Sheila S Teves, Luye An, Anders S Hansen, Liangqi Xie, Xavier Darzacq, Robert Tjian (2016) A dynamic mode of mitotic bookmarking by transcription factors eLife 5:e22280. https://doi.org/10.7554/eLife.22280 Sheila S Teves, Luye An, Aarohi Bhargava-Shah, Liangqi Xie, Xavier Darzacq, Robert Tjian (2018) A stable mode of bookmarking by TBP recruits RNA polymerase II to mitotic chromosomes eLife 7:e35621. https://doi.org/10.7554/eLife.35621 Kwan, J. Z. J., Nguyen, T. F., Uzozie, A. C., Budzynski, M. A., Cui, J., Lee, J. M. C., Van Petegem, F., Lange, P. F., & Teves, S. S. (2023). RNA Polymerase II transcription independent of TBP in murine embryonic stem cells. eLife, 12, e83810. https://doi.org/10.7554/eLife.83810 Price, R. M., Budzyński, M. A., Shen, J., Mitchell, J. E., Kwan, J. Z. J., & Teves, S. S. (2023). Heat shock transcription factors demonstrate a distinct mode of interaction with mitotic chromosomes. Nucleic acids research, 51(10), 5040–5055. https://doi.org/10.1093/nar/gkad304   Related Episodes In Vivo Nucleosome Structure and Dynamics (Srinivas Ramachandran) From Nucleosome Structure to Function (Karolin Luger) Structural Analysis of Nucleosomes During Transcription (Lucas Farnung)   Contact Epigenetics Podcast on Twitter Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Active Motif on Twitter Active Motif on LinkedIn Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Karim-Jean Armache from New York University - Grossman School of Medicine to talk about his work on the structural analysis of Polycomb Complex Proteins. Karim-Jean Armache started his research career with the structural characterization of the 12-subunit RNA Polymerase II. After starting his own lab he used this knowledge in x-ray crystallography and electron microscopy to study how gene silencing complexes like the PRC complex act on chromatin and influence transcription. Further work in the Armache Lab focused on Dot, a  histone H3K79 methyltransferase, and how it acts on chromatin, as well as how it is regulated by Histone-Histone crosstalk. References Armache, K. J., Garlick, J. D., Canzio, D., Narlikar, G. J., & Kingston, R. E. (2011). Structural basis of silencing: Sir3 BAH domain in complex with a nucleosome at 3.0 Å resolution. Science (New York, N.Y.), 334(6058), 977–982. https://doi.org/10.1126/science.1210915 Lee, C. H., Holder, M., Grau, D., Saldaña-Meyer, R., Yu, J. R., Ganai, R. A., Zhang, J., Wang, M., LeRoy, G., Dobenecker, M. W., Reinberg, D., & Armache, K. J. (2018). Distinct Stimulatory Mechanisms Regulate the Catalytic Activity of Polycomb Repressive Complex 2. Molecular cell, 70(3), 435–448.e5. https://doi.org/10.1016/j.molcel.2018.03.019 De Ioannes, P., Leon, V. A., Kuang, Z., Wang, M., Boeke, J. D., Hochwagen, A., & Armache, K. J. (2019). Structure and function of the Orc1 BAH-nucleosome complex. Nature communications, 10(1), 2894. https://doi.org/10.1038/s41467-019-10609-y Valencia-Sánchez, M. I., De Ioannes, P., Wang, M., Truong, D. M., Lee, R., Armache, J. P., Boeke, J. D., & Armache, K. J. (2021). Regulation of the Dot1 histone H3K79 methyltransferase by histone H4K16 acetylation. Science (New York, N.Y.), 371(6527), eabc6663. https://doi.org/10.1126/science.abc6663   Related Episodes Transcription and Polycomb in Inheritance and Disease (Danny Reinberg) From Nucleosome Structure to Function (Karolin Luger) Oncohistones as Drivers of Pediatric Brain Tumors (Nada Jabado)   Contact Epigenetics Podcast on Twitter Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Active Motif on Twitter Active Motif on LinkedIn eMail: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Akis Papantonis from the University Medical Center Göttingen to talk about his work on genome organisation mediated by RNA Polymerase II. The research of the Papantonis Laboratory focuses on investigating how environmental signalling stimuli are integrated by chromatin to control homeostatic to deregulated functional transitions. In more detail, the team is interested in how dynamic higher-order regulatory networks are influenced by the underlying linear DNA fiber. The ultimate goal of the laboratory is to understand general rules governing transcriptional and chromatin homeostasis and finally, how those rules might affect development, ageing or malignancies.   References Larkin, J. D., Cook, P. R., & Papantonis, A. (2012). Dynamic reconfiguration of long human genes during one transcription cycle. Molecular and cellular biology, 32(14), 2738–2747. https://doi.org/10.1128/MCB.00179-12 Diermeier, S., Kolovos, P., Heizinger, L., Schwartz, U., Georgomanolis, T., Zirkel, A., Wedemann, G., Grosveld, F., Knoch, T. A., Merkl, R., Cook, P. R., Längst, G., & Papantonis, A. (2014). TNFα signalling primes chromatin for NF-κB binding and induces rapid and widespread nucleosome repositioning. Genome biology, 15(12), 536. https://doi.org/10.1186/s13059-014-0536-6 Sofiadis, K., Josipovic, N., Nikolic, M., Kargapolova, Y., Übelmesser, N., Varamogianni-Mamatsi, V., Zirkel, A., Papadionysiou, I., Loughran, G., Keane, J., Michel, A., Gusmao, E. G., Becker, C., Altmüller, J., Georgomanolis, T., Mizi, A., & Papantonis, A. (2021). HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence. Molecular systems biology, 17(6), e9760. https://doi.org/10.15252/msb.20209760 Enhancer-promoter contact formation requires RNAPII and antagonizes loop extrusion. Shu Zhang, Nadine Übelmesser, Mariano Barbieri, Argyris Papantonis. bioRxiv 2022.07.04.498738; doi: https://doi.org/10.1101/2022.07.04.498738   Related Episodes Chromatin Organization During Development and Disease (Marieke Oudelaar) Biophysical Modeling of 3-D Genome Organization (Leonid Mirny) Hi-C and Three-Dimensional Genome Sequencing (Erez Lieberman Aiden)   Contact Epigenetics Podcast on Twitter Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Active Motif on Twitter Active Motif on LinkedIn Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Jane Mellor from the University of Oxford to talk about her work on H3K4me3, SET proteins, Isw1 and their role in transcription. Since the beginning of the century, Jane Mellor and her team have focused on H3K4 trimethylation and the factors that influence this mark. They discovered that H3K4me3 is an almost universal mark of the first nucleosome in every transcribed unit and all organisms. She could subsequently, together with the Kouzarides lab, identify SetD1, the enzyme that is responsible for writing this modification. Later on, the team characterized Isw1, a chromatin remodeler which “reads” H3K4me3. More recently the lab focuses on how the polymerase transcribes throughout the first nucleosomes of the transcribed region at the +2 nucleosome, with the help of Spt4.   References Santos-Rosa, H., Schneider, R., Bannister, A. J., Sherriff, J., Bernstein, B. E., Emre, N. C. T., Schreiber, S. L., Mellor, J., & Kouzarides, T. (2002). Active genes are tri-methylated at K4 of histone H3. Nature, 419(6905), 407–411. https://doi.org/10.1038/nature01080 Morillon, A., O'Sullivan, J., Azad, A., Proudfoot, N., & Mellor, J. (2003). Regulation of Elongating RNA Polymerase II by Forkhead Transcription Factors in Yeast. Science, 300(5618), 492–495. https://doi.org/10.1126/science.1081379 Morillon, A., Karabetsou, N., O'Sullivan, J., Kent, N., Proudfoot, N., & Mellor, J. (2003). Isw1 Chromatin Remodeling ATPase Coordinates Transcription Elongation and Termination by RNA Polymerase II. Cell, 115(4), 425–435. https://doi.org/10.1016/S0092-8674(03)00880-8 Uzun, Ü., Brown, T., Fischl, H., Angel, A., & Mellor, J. (2021). Spt4 facilitates the movement of RNA polymerase II through the +2 nucleosomal barrier. Cell Reports, 36(13), 109755. https://doi.org/10.1016/j.celrep.2021.109755   Related Episodes Effects of Non-Enzymatic Covalent Histone Modifications on Chromatin (Yael David) Nutriepigenetics: The Effects of Diet on Behavior (Monica Dus) Epigenetic Origins Of Heterogeneity And Disease (Andrew Pospisilik)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.21.349365v1?rss=1 Authors: Kearns, S., Mason, F. M., Rathmell, W. K., Park, I. Y., Walker, C., Verhey, K. A., Cianfrocco, M. A. Abstract: Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. A recently identified tubulin modification, methylation, occurs on mitotic spindle microtubules during cell division, and is enzymatically added to tubulin by the histone methyltransferase SETD2. We used a truncated version of human SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2 Rpb1 interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 has a higher activity towards tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrate that methylation is restricted to lysine 40 (K40) of -tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain, R1625C, bound to tubulin but could not methylate it whereas a mutation in the SRI domain, R2510H, caused loss of both tubulin binding and methylation. We thus further probed a role for the SRI domain in substrate binding and found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus RNA Polymerase II substrates, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Lastly, we found that substrate recognition also requires the negatively-charged C-terminal tail of -tubulin. Together, this work provides a framework for understanding how SETD2 serves as a dual methyltransferase for histone and tubulin methylation. Copy rights belong to original authors. Visit the link for more info

Wed, 30 Sep 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19068/ https://edoc.ub.uni-muenchen.de/19068/1/Niesser_Juergen_G.pdf Niesser, Jürgen Gerd ddc:540, ddc:500, Fakult

Tue, 21 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18520/ https://edoc.ub.uni-muenchen.de/18520/1/Muehlbacher_Wolfgang.pdf Mühlbacher, Wolfgang ddc:54

Gene transcription is a fundamental process of the living cell. Eukaryotic transcription of messenger RNA requires the regulated recruitment of the conserved transcribing enzyme RNA polymerase (Pol) II to the gene promoter. The most heavily regulated step is transcription initiation that involves the ordered assembly of Pol II, the general transcription factors (TF) -IIA, -IIB, -IID, -IIF, -IIE, -IIH and the co-activator Mediator complex. Mediator communicates between transcription regulators and Pol II, and is associated with human disease. Mediator from the yeast Saccharomyces cerevisiae (Sc) has a molecular mass of 1.4 megadaltons and contains 25-subunits that constitute a head, middle, tail and kinase module. The core of Mediator contains the head and middle modules that are essential for viability in Sc, and directly contact Pol II. Mediator co-operates with TFIIH, to assist assembly and stabilization of the transcription initiation complex and stimulate TFIIH kinase activity. Because of the large size and complexity of Mediator and the initiation machinery, the underlying mechanism remains poorly understood. In this work we studied the structure and function of Mediator head and middle modules, the structure of the reconstituted Pol II-core Mediator transcription initiation complex, and reveal mechanisms of transcription regulation. We report the crystal structure of the 6-subunit Schizosaccharomyces pombe Mediator head module at 3.4 Å resolution. The structure resembles the head of a crocodile and reveals eight elements that are part of three domains named neck, fixed jaw and movable jaw. The neck contains a spine, shoulder, arm and finger. The arm and essential shoulder elements contact the remainder of Mediator and Pol II. The head module jaws and central joint, important for transcription, also interact with Mediator and Pol II. The Sp head module structure is conserved and revises a 4.3 Å model of the Sc head module, explains known mutations, and provides an atomic model for one half of core Mediator. We further propose a model of the Mediator middle module based on protein crosslinking and mass spectrometry. To determine how Mediator regulates initiation, we prepared recombinant Sc core Mediator by co-expression of its 15 subunits in bacteria. Core Mediator is active in transcription assays and bound an in vitro reconstituted core initially transcribing complex (cITC) that contains Pol II, the general factors TFIIB, TBP, TFIIF, and promoter DNA. We determined the cryo-electron microscopy structure of the initially transcribing core initiation complex at 7.8 Å resolution. The structure reveals the arrangement of DNA, TBP, TFIIB, and TFIIF on the Pol II surface, the path of the complete DNA template strand and three TFIIF elements. The ‘charged helix’ and ‘arm’ of TFIIF subunit Tfg1, reach into the Pol II cleft and may stabilize open DNA. The linker region of TFIIF subunit Tfg2 extends between Pol II protrusion and TFIIB, and may stabilize TFIIB. The structure agrees with its human counterpart, and suggests a conserved architecture of the core initiation complex. Finally, we determined the cryo-electron microscopy architecture of the cITC-core Mediator complex at 9.7 Å resolution. Core Mediator binds Pol II at the Rpb4/Rbp7 stalk close to the carboxy-terminal domain (CTD). The Mediator head module contacts the Pol II dock and TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module ‘plank’ domain touches the Pol II foot and may control polymerase conformation allosterically. The Med14 subunit bridges head and middle modules with a ‘beam’, and connects to the tail module that binds transcription activators located on upstream DNA. The ‘arm’ and ‘hook’ domains of core Mediator form part of a ‘cradle’ that may position CTD and the TFIIH kinase to stimulate Pol II phosphorylation. Taken together, our results provide a structural framework to unravel the role of Mediator in transcription initiation and determine mechanisms of gene regulation.

Mon, 20 Jan 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17932/ https://edoc.ub.uni-muenchen.de/17932/1/Hoeg_Friederike.pdf Hög, Friederike

Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II). This process is tightly regulated and coupled to RNA processing. Many transcription and RNA processing factors are recruited to Pol II via its conserved C-terminal domain (CTD) containing 27 heptapeptide repeats of the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 in Saccharomyces cerevisiae. These repeats can be differentially phosphorylated during the transcription cycle serving as a code for interacting factors. During transcription initiation, Ser5 is phosphorylated at the 5’-end of genes and this phosphorylation is required for RNA capping enzyme binding. During transcription elongation, the Pol II CTD becomes phosphorylated at Tyr1 and Ser2 and binds the elongation factor Spt5. Spt5 also contains a repetitive C-terminal region (CTR) required for cotranscriptional recruitment of proteins. At the 3’-end of genes, Ser2-phosphorylated Pol II associates with the cleavage and polyadenylation factor (CPF) and is dephosphorylated at Tyr1 residues. This work shows that CPF is a Pol II CTD phosphatase and that its subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, Glc7 activity is required for normal Tyr1 dephosphorylation at the polyadenylation (pA) site, for recruitment of termination factors Pcf11 and Rtt103, and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing and transcription termination are coupled via CPF-dependent Pol II Tyr1 dephosphorylation. Additionally, 19 kinases were tested for activity on Tyr1 in yeast by selective inhibition or knock-out in vivo. However, none of the candidates was identified as the Tyr1 kinase. Possibly this enzyme is an atypical kinase not known to be involved in transcription so far. Furthermore, this work reports a new role of the Spt5 CTR in recruitment of RNA 3’-end processing factors. The results show that the Spt5 CTR as well as RNA is required for normal recruitment of the pre-mRNA cleavage factor (CF) I to the 3’-end of yeast genes. Genome-wide ChIP profiling detects occupancy peaks of CFI subunits around 100 base pairs downstream of the pA site of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at Ser2 of the CTD. Consistent with this model, the CTR interacts with CFI in vitro, but is not required for pA site recognition and transcription termination in vivo. In summary, we characterized two new aspects of transcription and RNA processing regulation by two different C-terminal repetitive protein domains. CTD Tyr1 phosphorylation, which is removed by Glc7, regulates termination factor recruitment by masking their binding site, the Spt5 CTR is involved in recruitment of CFI. Both results greatly contribute to a more detailed understanding of the mechanisms involved in transcription termination and RNA 3’-end processing.

X chromosome inactivation (XCI) in female mammalian cells is an ideal model system to study the relationship of epigenetic regulation and higher-order chromatin structure. However, light microscopic studies of chromosomal organization have long been limited by the diffraction barrier of optical resolution. Super-resolution 3D-structured illumination microscopy (3D-SIM) – one of several recent techniques that circumvent this limitation – enables multicolor optical sectioning of entire cells with eightfold-improved volumetric resolution compared to conventional fluorescence imaging methods. In the present work, 3D-SIM has been applied to analyze higher-order chromatin structure of the Barr body in mammalian nuclei, a characteristic hallmark of XCI, with yet unprecedented detail. First, the increased resolution prompted to reappraise the potential detrimental effect of the DNA-FISH procedure on chromatin structure. Comparative analyses revealed slight deteriorations at the resolution level of 3D-SIM, especially within more decondensed euchromatin sites within the nuclear interior. In contrast, overall nuclear morphology and the nuclear envelope as well as heterochromatic sites in general maintained well preserved. The results suggest that DNA-FISH studies can benefit from a combination with super-resolution microscopy. In particular, when keeping in mind the current developments of the FISH technique with increasingly small and higher-complexity probes. The compact shape of the Barr body led to the assumption of a contribution of this special higher-order chromatin structure to the establishment and maintenance of the silenced state in the inactive X chromosome (Xi). However, a confirmation of this view has always been hampered by the restrictions of conventional light microscopy. In this work, the 3D chromosomal organization of the Xi and autosomes has been investigated with 3D-SIM in various human and mouse somatic cells and in mouse embryonic stem cell (ESC) lines. The precise subchromosomal localization of a variety of factors involved in XCI in different developmental states was qualitatively and quantitatively assessed utilizing combined immunofluorescence, EdU- pulse and RNA-/DNA-FISH labeling protocols and novel data analysis tools customized for the special requirements of 3D-SIM. The results demonstrate that all autosomes are made of a three-dimensional interconnected network of chromatin domains (CDs, or topology associated domains, TADs) of highly-variable shape and dynamics. CDs/TADs are comprised of a compacted chromatin core enriched with repressive marks, which is collectively proposed to be the functionally passive chromatin compartment (PNC). This PNC is surrounded by a 50 – 150 nm locally defined, less compacted perichromatin region (PR) that is enriched with active histone modifications and pervaded by a three-dimensional interchromatin (IC) network. The PR and the IC are collectively referred to as being the functionally relevant active nuclear compartment (ANC) that harbors all major nuclear processes, including transcription and replication. 3D-SIM data revealed that the Barr body maintains this principle compartmentalization and that it is still pervaded by a narrow ANC network, which is able to fulfill its functional role as a hub for replication or rarely occurring expression of XCI-escape genes. Live-cell super-resolution imaging on HeLa H2B-GFP cells confirmed that the observed chromatin features do not reflect fixation artifacts. Xist RNA, the key factor of XCI, has been found to be preferentially located as distinct discernible foci within the ANC throughout the entire volume of the Barr body. Here, it is tightly associated with a Xi-specific form of the nuclear matrix protein SAF-A, which confirms a previously suggested role for this Xi-enriched protein in Xist RNA spreading. In contrast, Xist RNA shows no spatial correlation with repressive Xi-enriched histone marks that are found within compacted chromatin sites. This specific localization of Xist RNA reflects an intrinsic feature as it is already present during early spreading in differentiating female ESCs, where it precedes chromatin compaction concomitant with RNA Polymerase II exclusion. Its localization is further confirmed in a male ESC line carrying an inducible Xist transgene on an autosome, but where Xist RNA fails to form a true autosomal Barr body, which is less compacted and maintains transcriptional activity. Last, Xist RNA shows no direct association with PRC2, the mediator of H3K27me3, which is in contrast to the generally believed direct recruitment model of PRC2 to the Xi by Xist RNA. The data collected in this work reflects further support and a refinement of the not unequivocally accepted CT-IC (chromosome territory - interchromatin compartment) model of higher-order chromosome architecture. In addition, a first attempt has been made to integrate these findings with a recently growing number of studies using chromosome conformation capturing (3C)-based techniques and to complement them on the single-cell level. Finally, a novel model for Xist RNA function in XCI is presented, which proposes a sequence-independent structural role for gene silencing and the formation of a repressive chromatin compartment.

Mon, 14 Oct 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16181/ https://edoc.ub.uni-muenchen.de/16181/1/Kinkelin_Kerstin.pdf Kinkelin, Kerstin ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Tue, 3 Sep 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16965/ https://edoc.ub.uni-muenchen.de/16965/1/Lidschreiber_Michael.pdf Lidschreiber, Michael Maximilian ddc:540, ddc:500, Fakultät

Fri, 8 Feb 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16703/ https://edoc.ub.uni-muenchen.de/16703/1/Burger_Kaspar.pdf Burger, Kaspar ddc:570, ddc:500, Fakultät für Biolog

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

Thu, 24 May 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15132/ https://edoc.ub.uni-muenchen.de/15132/1/Hintermair_Corinna.pdf Hintermair, Corinna

Mon, 4 Apr 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13567/ https://edoc.ub.uni-muenchen.de/13567/1/Seizl_Martin.pdf Seizl, Martin Josef ddc:540, ddc:500, Fakultät für Chemie

Thu, 10 Feb 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12769/ https://edoc.ub.uni-muenchen.de/12769/1/Czeko_Elmar.pdf Czeko, Elmar ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 31 Jan 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14013/ https://edoc.ub.uni-muenchen.de/14013/1/Lehmann_Elisabeth.pdf Lehmann, Elisabeth ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Transcription of protein coding genes by RNA polymerase II (RNAPII) is an essential step in gene expression. Transcription elongation is a highly dynamic and discontinuous process that includes frequent pausing of RNAPII, backtracking, and arrest both in vitro and in vivo. Consequently, a multitude of transcription elongation factors are needed for efficient transcription elongation. When transcription elongation factors fail to “restart” RNAPII the persistently stalled RNAPII complex prevents transcription and thus has to be recognized and removed to free the gene for subsequent polymerases. Similarly, DNA damage causes stalling of RNAPII. In this case, the DNA damage is either repaired by Transcription-Coupled Repair (TCR) or RNAPII is degraded as a “last resort” mechanism by the ubiquitin proteasome system. In contrast to RNAPII degradation caused by DNA damage, the cellular pathway for removal of transcriptionally stalled RNAPII complexes has remained largely obscure. However, it was speculated that transcriptionally stalled RNAPII complexes are degraded by the same pathway as RNAPII stalled due to DNA damage. Here, it is shown that the pathway for degradation of transcriptionally stalled RNAPII is distinct from the DNA damage-dependent pathway, providing the first evidence that the cell distinguishes between RNAPII complexes stalled for different reasons. The novel cellular pathway for transcriptional stalling-dependent degradation of RNAPII is termed TRADE. Specifically, in the TRADE pathway a different yet overlapping set of enzymes is responsible for poly- and de-ubiquitylation of transcriptionally stalled RNAPII. Moreover, the catalytic 20S proteasome is recruited to transcribed genes indicating that Rpb1 of transcriptionally stalled RNAPII complexes is degraded at the site of transcription. Importantly, nucleotide starvation and temperature stress which might mimic natural conditions of transcription elongation impairment also lead to RNAPII degradation. Finally, this study provides the first evidence that the mechanism for the controlled degradation of the transcriptionally stalled RNA polymerase complex might also exist for transcription by RNAPI and RNAPIII. Taken together, the TRADE pathway elucidated in this study ensures continued transcription.

Thu, 22 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12021/ https://edoc.ub.uni-muenchen.de/12021/1/Heidemann_Martin.pdf Heidemann, Martin

Fri, 17 Jul 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11254/ https://edoc.ub.uni-muenchen.de/11254/1/Andrecka_Joanna.pdf Andrecka, Joanna ddc:540, ddc:500, Fakultät für Chemie und

Archaea combine bacterial with eukaryotic features to regulate cellular processes. While initiation of transcription resembles the eukaryotic RNA-Polymerase II apparatus, transcriptional regulation is predominantly bacteria-like. In this work the gene expression profile of amino acid metabolism and metal dependent processes in Halobacterium salinarum R1 was elucidated. To gain insights into transcriptional regulatory processes, microarray technology was used as a global approach. Genes encoding certain DNA binding proteins were deleted and/or overexpressed, to compare the expression pattern of the deletion- and overexpression strains, respectively, with the parental strain R1. For a better understanding of metal dependent processes H. salinarum was grown under iron starvation and compared to cells grown under normal conditions. For the investigation of metal dependent processes the DNA binding proteins SirR and TroR were chosen. To determine a possible function of the regulator protein, conclusions were drawn from a comparison of the deletion mutants ∆sirR and ∆troR, respectively, with the parental strain. SirR (staphylococcal iron regulator repressor) was shown to repress the expression of a Fe(II)/Mn(II) dependent ABC-transporter in the presence of iron. In accordance with this data the same transport operon was shown to be induced under iron starvation. Furthermore, TroR (transport related operon) was shown to repress the expression of a Mn(II)-dependent ABC-transporter. In addition, TroR induces the gene expression of the metal dependent regulator gene idr2, which represses together with iron siderophor synthesis genes. To study the amino acid metabolism in H. salinarum Lrp (leucine-responsive regulatory protein) proteins were chosen, because in both archaea and bacteria Lrp is connected to the coordination of amino acid metabolism. To take a closer look on Lrp-homologs further investigation were performed with lrp and lrpA1. Both genes are located next to genes, encoding proteins involved in amino acid metabolism. Possible Lrp target genes were identified by either constructing lrp and lrpA1 deletion mutants or overexpressing the two genes. Microarray analysis revealed that Lrp functions as a global regulator of transcription. Lrp activates the gene expression of the glutamine synthetase gene glnA, regulates the peptide- and phosphate transport, as well as the central intermediary metabolism, and activates the expression of the transcriptional regulator sirR. By the control of sirR gene expression through Lrp correlation between amino acid metabolism and metal dependent processes could be demonstrated. In contrast to Lrp, LrpA1 regulates gene expression of less genes, amongst them the aspartate transaminase gene aspB3, so that further studies were focussed on the gene regulation of aspB3. The second part of this work examines with specific protein-DNA interactions. Prior to interaction studies, RACE-analysis was used to determine 5´UTR and 3´UTR of certain transcripts. To perform protein-DNA binding studies LrpA1 and TroR were recombinantly expressed in Escherichia coli. A DNA-binding assay adapted to halophilic conditions revealed manganese dependent binding of TroR to its own promoter region. LrpA1 was also shown to bind to the lrpA1 promoter region, as well as an aspartate dependent binding to the aspB3 promoter region. CD-spectroscopy experiments could prove that the interaction between L-aspartate and LrpA1 stabilizes the secondary structure of the protein. To gain more insights into the LrpA1 and L-aspartate dependent aspB3 gene expression, northern blot analysis were performed, that showed an induction of the aspB3 transcription in the absence of L- aspartate. This occurs either in a medium lacking aspartate or after aspartate is metabolized in the stationary phase. At the same time, an induction of the lrpA1 gene expression was observed. This can be illustrated in a model that postulates a reciprocal regulation of the lrpA1 and aspB3 gene expression.

Wed, 14 Jan 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/9698/ https://edoc.ub.uni-muenchen.de/9698/1/Jasiak_Anna_Justyna.pdf Jasiak, Anna Justyna ddc

RNA polymerase II (RNAPII) has been identified almost 40 years ago, but the molecular details of its regulation and fine tuning during messenger RNA (mRNA) synthesis are still far from understood. Subsequently to RNAPII six general transcription factors (GTFs; TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH) were discovered of which all except TFIIA are necessary and sufficient for promoter-dependent basal transcription initiation. In addition to the GTFs activator-dependent transcription requires the presence of a transcription cofactor, the Mediator complex. Mediator serves as a link between transcription activators, enhancers and the general transcription machinery. Initial studies revealed that Mediator stimulates the activity of the TFIIH associated kinase CDK7 and thereby facilitates RNAPII C-terminal domain (CTD) phosphorylation. Furthermore the Mediator complex interacts functionally with several signal transduction pathways and serves as an signal integration platform. In order to dissect the process of transcription initiation, early studies made use of in vitro transcription systems reconstituted from recombinant or highly purified GTFs and RNAPII. In this system basal, activator-independent transcription does not require the presence of the Mediator complex. If however a more physiological nuclear extract transcription system is used, our laboratory and others have established previously that basal transcription becomes critically dependent on Mediator. Another difference between both transcription systems is that the first is insensitive to the kinase inhibitor H8 whereas in the second transcription can be inhibited by H8. This suggests that only the second transcription system is regulated by RNAPII CTD phosphorylation. In this thesis the interplay between Mediator, RNAPII, GTFs and transcription cofactors was studied using immobilized promoter template assays in combination with various immunodepleted nuclear extracts and recombinant factors. Negative cofactor 2 (NC2) is an evolutionary conserved general cofactor that binds to many active genes in vivo. Previous studies in our laboratory had shown with recombinant proteins that NC2 competes with TFIIA and TFIIB for binding to TATA-binding protein (TBP) at a promoter in vitro. Genetic studies in yeast provided evidence that Mediator acts antagonistically to NC2. Here I have studied the role of NC2 on preinitiation complex (PIC) formation and transcription in nuclear extracts. I observed rapid association of TFIID with promoters whereas NC2 enters PICs with a slow kinetic which is similar to that of TFIIB recruitment. My data indirectly suggest that TBP binds to DNA in a yet to be defined inactive form (perhaps as a TFIID complex) which is then slowly converted into an active TBP-TATA complex that is rapidly recognized by GTFs or NC2. My data support the notion that NC2 and TFIIB compete for binding to a PIC also in immobilized promoter assays under physiological conditions. NC2 concentrations in nuclear extracts appears to be tightly controlled. Doubling the NC2 concentration in a nuclear extract by adding recombinant NC2 (rNC2) abolished functional PIC formation and transcription. However, the in vitro analysis also showed that upstream of NC2 PIC formation is fully dependent on Mediator. Hence, TFIID binds to a promoter in a nuclear extract in vitro transcription system but we have no indication that a transcription competent PIC is formed in the absence of Mediator. In yeast studies it was reported that upon transcription initiation in vitro several GTFs dissociate from the promoter DNA template whereas the Mediator complex is retained in a reinitiation complex. In the human system I recapitulate this observation for TFIIB and CDK7. In addition I provide evidence that Mediator partially dissociated from the promoter template upon transcription initiation. Upon transcription initiation the middle module subunit MED7 was retained on a promoter template, whereas the tail module subunit MED15 and CDK8 did dissociate. This data suggest that upon transcription initiation a head/middle module Mediator subcomplex is retained at the promoter whereas the tail and CDK8 modules dissociate. Previous studies have established that Mediator promotes CDK7-dependent phosphorylation of the RNAPII CTD at serine-5 (ser-5). Various studies found that CTD ser-5 phosphorylation does coincide with transcription initiation. Using new monoclonal antibodies I observed two functionally distinct modes of CTD ser-5 phosphorylation in vitro: Hypo- and hyperphosphorylation of the largest RNAPII subunit Rpb1. I observed that CTD ser-5 hypophosphorylation is established already before complex opening by TFIIH. I found CTD ser-5 hypophosphorylation to be critically dependent on TBP, Mediator, TFIIB and CDK7. In addition I noted that CTD ser-5 hypophosphorylation correlates with the transcription potential of a PIC. CTD ser-5 hyperphosphorylation was established in a Mediator-dependent fashion but independent of productive transcription. Immunodepletion of CDK7 did not led to a reduction in CTD ser-5 hyperphosphorylation. However, immunodepletion of CDK8 caused a reduction but not a loss of CTD ser-5 hyperphosphorylation upon transcription initiation indicating that another yet to be identified kinase might be involved in this process. These data suggest that CTD ser-5 hypophosphorylation is established only in the PIC context on RNAPII located at bona fide promoter regions but not on RNAPII complexes bound to DNA outside of promoter regions, e.g. in an open reading frame. Recently phosphorylation of the RNAPII CTD at serine-7 (ser-7) was reported. In that study the entire coding region of the TCRβ locus was found to be associated with RNAPII CTD phosphorylated at ser-7. Starting from there I found that establishment of CTD ser-7 phosphorylation in the process of transcription initiation can be recapitulated in an immobilized template assay system in vitro. I confirmed the in vitro finding that establishment of CTD ser-7 phosphorylation correlates with transcription initiation with chromatin immunoprecipitation experiments on an inducible model gene system in vivo. Similar to CTD ser-5 phosphorylation, I observed two modes of CTD ser-7 phosphorylation: CTD ser-7 hypo- and hyperphosphorylation. In contrast to CTD ser-5 hypophosphorylation, which was established before complex opening, I observed establishment of CTD ser-7 hypophosphorylation predominantly after complex opening by TFIIH. Both, CTD ser-7 hypo- and hyperphosphorylation were found to be Mediator-dependent. A mass spectrometric screen for PIC associated kinases (in collaboration with the laboratory of Gerhard Mittler) yielded 13 kinases. Seven of the identified kinases were further tested for their potential to phosphorylate the RNAPII at ser-7 in an immobilized template assay.

RNA polymerase II (Pol II) is the eukaryotic enzyme responsible for transcribing all protein-coding genes into messenger RNA (mRNA). This thesis describes studies on the molecular mechanisms of Pol II translocation, alpha-amanitin inhibition and DNA lesion recognition by Pol II. To study how Pol II translocates after nucleotide incorporation, we prepared elongation complex (EC) crystals in which pre- and post-translocation states interconvert. Crystal soaking with the inhibitor alpha-amanitin locked the EC in a new state that we identified as a translocation intermediate at 3.4 Å resolution. The DNA base entering the active site occupies a “pre-templating” position above the central bridge helix, which is shifted and occludes the standard templating position. A leucine residue in the trigger loop forms a wedge next to the shifted bridge helix, but moves by 13 Å to close the active site for nucleotide incorporation. Our results support a Brownian ratchet mechanism of elongation that involves swinging of the trigger loop between open, wedged, and closed positions, and suggest that alpha-amanitin impairs nucleotide incorporation and translocation by trapping the trigger loop and bridge helix in a translocation intermediate. Cells use transcription-coupled repair (TCR) to efficiently eliminate DNA lesions such as UV-induced cyclobutane pyrimidine dimers (CPDs). Here we present the structure-based mechanism for the first step in eukaryotic TCR, CPD-induced stalling of Pol II. A CPD in the transcribed strand slowly passes a translocation barrier, and enters the polymerase active site. The CPD 5’-thymine then directs uridine monophosphate (UMP) misincorporation into mRNA, which blocks translocation. Artificial replacement of the UMP by adenosine monophosphate (AMP) enables CPD bypass, thus Pol II stalling requires CPD-directed misincorporation. In the stalled complex, the lesion is inaccessible, and the polymerase conformation is unchanged. This is consistent with non-allosteric recruitment of repair factors and excision of a lesion-containing DNA fragment in the presence of Pol II. CPD recognition is compared with the recognition of a cisplatin-induced guanine-guanine intrastrand crosslink. Similarities and differences in the detailed mechanism of transcriptional stalling at the two different dinucleotide lesions are discussed.

DNA lesions arising from environmental and endogenous sources induce various cellular responses including cell cycle arrest, DNA repair and apoptosis. Although detailed insights into the biochemical mechanisms and composition of DNA repair pathways have been obtained from in vitro experiments, a better understanding of the interplay and regulation of these pathways requires DNA repair studies in living cells. In this study we employed laser microirradiation and photobleaching techniques in combination with specific mutants and inhibitors to analyze the real-time accumulation of proteins at laser-induced DNA damage sites in vivo, thus unravelling the mechanisms underlying the coordination of DNA repair in living cells. The immediate and faithful recognition of DNA lesions is central to cellular survival, but how these lesions are detected within the context of chromatin is still unclear. In vitro data indicated that the DNA-damage dependent poly(ADP-ribose) polymerases, PARP-1 and PARP-2, are involved in this crucial step of DNA repair. With specific inhibitors, mutations and photobleaching analysis we could reveal a complex feedback regulated mechanism for the recruitment of the DNA damage sensor PARP-1 to microirradiated sites. Activation of PARP-1 results in localized poly(ADP-ribosyl)ation and amplifies a signal for the subsequent rapid recruitment of the loading platform XRCC1 which coordinates the assembly of the repair machinery. Using similar techniques we could demonstrate the immediate and transient binding of the RNA Polymerase II cofactor PC4 to DNA damage sites, which depended on its single strand binding capacity. This establishes an interesting link between DNA repair and transcription. We propose a role for PC4 in the early steps of the DNA damage response, recognizing and stabilizing single stranded DNA (ssDNA) and thereby facilitating DNA repair by enabling repair factors to access their substrates. After DNA lesions have been successfully detected they have to be handed over to the repair machinery which restores genome integrity. Efficient repair requires the coordinated recruitment of multiple enzyme activities which is believed to be controlled by central loading platforms. As laser microirradiation induces a variety of different DNA lesions we could directly compare the recruitment kinetics of the two loading platforms PCNA and XRCC1 which are involved in different repair pathways side by side. We could demonstrate that PCNA and XRCC1 show distinct recruitment and binding kinetics with the immediate and fast recruitment of XRCC1 preceding the slow and continuous recruitment of PCNA. Introducing consecutively multiple DNA lesions within a single cell, we further demonstrated that these different recruitment and binding characteristics have functional consequences for the capacity of PCNA and XRCC1 to respond to successive DNA damage events. To further study the role of PCNA and XRCC1 as loading platforms in DNA repair, we extended our analysis to their respective interaction partners DNA Ligase I and III. Although these DNA Ligases are highly homologous and catalyze the same enzymatic reaction, they are not interchangeable and fulfil unique functions in DNA replication and repair. With deletion and mutational analysis we could identify domains mediating the specific recruitment of DNA Ligase I and III to distinct repair pathways through their interaction with PCNA and XRCC1. We conclude that this specific targeting may have evolved to accommodate the particular requirements of different repair pathways (single nucleotide replacement vs. synthesis of short stretches of DNA) and thus enhances the efficiency of DNA repair. Interestingly, we found that other PCNA-interacting proteins exhibit recruitment kinetics similar to DNA Ligase I, indicating that PCNA not only serves as a central loading platform during DNA replication, but also coordinates the recruitment of multiple enzyme activities to DNA repair sites. Accordingly, we found that the maintenance methyltransferase DNMT1, which is known to associate with replication sites through binding to PCNA, is likewise recruited to DNA repair sites by PCNA. We propose that DNMT1, like in DNA replication, preserves methylation patterns in the newly synthesized DNA, thus contributing to the restoration of epigenetic information in DNA repair. In summary, we found immediate and transient binding of repair factors involved in DNA damage detection and signalling, while repair factors involved in the later steps of DNA repair, like damage processing, DNA ligation and restoration of epigenetic information, showed a slow and persistent accumulation at DNA damage sites. We conclude that DNA repair is not mediated by binding of a preassembled repair machinery, but rather coordinated by the sequential recruitment of specific repair factors to DNA damage sites.

Gene expression encompasses a multitude of different steps, starting with transcription in the nucleus, co-transcriptional processing and packaging of the mRNA into a mature mRNP, export of the mRNP through the nuclear pore and finally the translation of the message in the cytoplasm. The central coordinator for coupling of the nuclear events is the differentially phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAP II). The phosphorylation pattern of the CTD not only dictates the progression through the transcription cycle but also the recruitment of mRNA processing machineries. Coupling of transcription to mRNA export is achieved by the TREX complex, which consists in the yeast S. cerevisiae of the heterotetrameric THO complex important for transcription elongation, the SR-like proteins Gbp2 and Hrb1, and Tex1 and the mRNA export factors Sub2 and Yra1. By direct interaction with Yra1, the mRNA export receptor Mex67-Mtr2 is then recruited to the mRNP and transports the mRNP through the nuclear pore complex to the cytoplasm. In a genetic screen for factors that are crucial for TREX complex function in the living cell, Ctk1, a cyclin dependent kinase (CDK) that phosphorylates the C-terminal domain (CTD) of RNAP II during transcription elongation, was identified (Hurt et al. 2004). Surprisingly, besides the TREX components Gbp2 and Hrb1, Ctk1 co-purified ribosomal proteins and translation factors. Using sucrose density centrifugation, it could be shown that Ctk1 indeed associates with translating ribosomes in vivo, suggesting a novel function of this protein in translation. This assumption was confirmed by in vitro translation assays: loss of Ctk1 function leads to a reduction in translational activity. More specifically, Ctk1 is important for efficient translation elongation and contributes to the accurate decoding of the message. Cells depleted for Ctk1 are more sensitive towards drugs that impair translational accuracy and show an increase in the frequency of miscoding in vivo. The function of Ctk1 during decoding of the message is most likely direct, as in extracts of cells depleted for Ctk1 the defect in correct decoding of the message can be restored to wild type levels by addition of purified CTDK-I complex. An explanation for the molecular mechanism of Ctk1’s function is provided by the identification of Rps2 as a novel substrate of Ctk1. Rps2 is a protein of the small ribosomal subunit, located at the mRNA entry tunnel and known to be essential for translational accuracy. Importantly, Rps2 is phosphorylated on serine 238 by Ctk1, and cells containing an rps2-S238A mutation show an increased sensitivity towards drugs that affect translational accuracy and an increase in miscoding as determined by in vitro translation extracts. The role of Ctk1 in translation is probably conserved as CDK9, the mammalian homologue of Ctk1, also associates with polysomes. Since Ctk1 interacts with the TREX complex, which functions at the interface of transcription and mRNA export, Ctk1 might bind to the mRNP during transcription and accompany the mRNP to the ribosomes, where Ctk1 enhances efficient and accurate translation of the mRNA. This study could be an example of a novel layer of control in gene expression: the composition of the mRNP determines its translational fate, including efficiency and accuracy of translation.

RNA polymerase II (RNAP II) transiently associates with many different proteins and multiprotein complexes during the mRNA transcription cycle, which includes three phases, initiation, elongation, and termination. This thesis describes structural studies of two factors that facilitate transcription through chromatin. The heterodimeric Saccharomyces cerevisiae elongation factor Spt4-Spt5 (human DSIF) has been identified by biochemical and genetic approaches to help RNAP II transcribe through chromatin. It is assumed that Spt4-Spt5 pauses RNAP II to open a time window for capping enzyme recruitment and addition of a cap to the 5'-end of the nascent RNA. The preparation of milligram quantities of soluble Spt4-Spt5 variants that are suited for structural studies has been achieved. Several strategies to resolve the structure of the RNAP II–Spt4-Spt5 complex were unsuccessful, possibly indicating an intrinsic flexibility of the complex. In addition, there is now evidence for direct links between chromatin modification and transcription elongation. A major player in this process is the histone lysine methyltransferase Set2 which has a modular structure. The catalytic activity of Set2 is mediated by the SET [Su(var)3-9, Enhancer of Zeste, Trithorax] domain. During mRNA elongation, the SRI (Set2 Rpb1-interacting) domain of Set2 binds to the phosphorylated CTD (carboxyl-terminal domain) of RNAP II. The NMR solution structure of yeast Set2 SRI domain has been determined. The structure reveals a novel CTD-binding fold consisting of a left-handed three-helix bundle. Unexpectedly, the SRI domain fold resembles the structure of an RNA polymerase-interacting domain in sigma factors that mediate transcription initiation in bacteria (domain sigma2 in sigma70). NMR titration experiments show that the SRI domain binds a Ser2/Ser5-phosphorylated CTD peptide comprising two heptapeptide repeats and three flanking NH2-terminal residues. Amino acid residues that show strong chemical shift perturbations upon CTD binding cluster in two regions on the SRI surface. The results will enable a detailed analysis of the specific CTD interactions underlying the coupling of transcription and chromatin modification by Set2.

TFIIF is the only general transcription factor that has been implicated in the preinitiation complex assembly, open complex formation, initiation and transcription elongation. In addition, TFIIF stimulates Fcp1, a central phosphatase needed for recycling of RNA polymerase II (Pol II) after transcription by dephosphorylation of the Pol II C-terminal domain (CTD). This thesis reports the X-ray structure of the small CTD phosphatase Scp1, which is homologous to the Fcp1 catalytic domain. The structure shows a core fold and an active center similar to phosphotransferases and –hydrolases that solely share a DXDX(V/T) signature motif with Fcp1/Scp1. It was further demonstrated that the first aspartate in the signature motif undergoes metalassisted phosphorylation during catalysis, resulting in a phosphoaspartate intermediate that was structurally mimicked with the inhibitor beryllofluoride. Specificity may result from CTD binding to a conserved hydrophobic pocket between the active site and an insertion domain that is unique to Fcp1/Scp1. Fcp1 specificity may additionally arise from phosphatase recruitment near the CTD via the Pol II subcomplex Rpb4/7, which is shown to be required for Fcp1 binding to the polymerase in vitro. Until now, the main impediment in the high resolution crystallographic studies of TFIIF in complex with Pol II and other members of transcription machinery was unavailability of soluble, stoichiometric TFIIF complex in sufficient amounts. This thesis reports on the development of the overexpression system in yeast and a purification protocol that enabled for the first time to isolate milligram amounts of a pure and soluble, 15-subunit (~0,7 MDa) stoichiometric Pol IITFIIF complex. Such complex together with the promoter DNA, RNA, TBP and TFIIB assembles in vitro into the yeast initially transcribing complex, which can now be studied structurally.

RNA polymerase II (Pol II) is the central enzyme, that synthetizes all mRNA in eukaryotic cells. In this work, I solved the structure of the complete, initiation-competent 12-subunit yeast RNA polymerase II at 3.8 Å. I also solved the structure of the Pol II subcomplex of Rpb4/7 alone at 2.3 Å resolution. These structures reveal the details of Pol II assembly from 12 subunits and give important insights into the initiation of transcription. The refined, atomic model of the complete 12-subunit Pol II enabled homology modeling of the two other nuclear RNA polymerases. In Pol I and Pol III, 65 % and 77 % of the Pol II fold are conserved, respectively. Together with a recent structure of a Pol II elongation complex, these results show that the basic mechanism of transcription applies also to the two other nuclear RNA polymerases

This thesis describes crystal structures of complete, 12-subunit yeast RNA polymerase II (Pol II) in complex with a synthetic transcription bubble and product RNA, with an NTP substrate analogue, and in complex with the transcription elongation factor TFIIS. The structure of the Pol II-transcription bubble-RNA complex reveals incoming template and non-template DNA, a seven base-pair DNA-RNA hybrid, and three nucleotides each of separating DNA and RNA. Based on this structure, those parts of Pol II were identified which are involved in separating template DNA from non-template DNA before the active site, and DNA from product RNA at the upstream end of the DNA-RNA hybrid. In both instances, strand separation can be explained by Pol II-induced duplex distortions. Only parts of the complete transcription bubble present in the complexes are ordered in the crystal structure, explaining the way in which high processivity of Pol II is reconciled with rapid translocation along the DNA template. The presence of an NTP substrate analogue in a conserved putative pre-insertion site was unveiled in a Pol II-transcription bubble-RNA complex crystal soaked with the substrate analogue GMPCPP. The structure of the Pol II-TFIIS complex was obtained from Pol II crystals soaked with TFIIS. TFIIS extends from the Pol II surface to the active site and complements the active site with two essential and invariant acidic residues for hydrolytic RNA cleavage. TFIIS also induces extensive structural changes in Pol II that reposition nucleic acids, in particular RNA, near the active centre. These results support the idea that Pol II contains a single tuneable active site for RNA polymerisation and cleavage. The technical obstacles imposed by crystal structure determination of large, transient protein-DNA-RNA complexes were overcome by two novel, fluorescence-based assays to monitor and optimise the composition of the crystals. Both assays are not limited to Pol II complexes, but can serve as a general tool for the crystallographic community.

Proteine leisten den entscheidenden Beitrag zur Struktur und Funktion der Zellen aller Lebewesen.Vieles spricht daher für eine Betrachtung des Proteoms oder einer Teilmenge davon (Subproteom), um den Zustand lebender Zellen zu beschreiben. Mit optimierten und neuen Methoden wurde das regulatorische Netzwerk, welches auf der Ebene der Trankription die Expression von Proteinen entscheidend mitbestimmt, genauer betrachtet. Hierzu wurden Zellfraktionierungsprotokolle optimiert, um die Proteine des Zellkerns und des Chromatins anzureichern und in nachfolgenden Analysen verwenden zu können. Es wurden Antikörper gegen verbreitete, funktional relevante Peptidmotive generiert. Aufbauend auf dem Konzept der Motivantikörper wurde eine Färbemethode für Zellkernproteine mit Kernlokalisationssignal entwickelt. Schließlich wurde mit den Methoden der Zellfraktionierung und spezifischeren Antikörpertechniken eine Analyse des humanen Mediator-Komplexes der RNA Polymerase II unternommen, die zur Entdeckung neuer Untereinheiten, potentiell interagierender Proteine und Phosphorylierungen im Komplex führte.

Doktorarbeit zur Untersuchung der grossen Untereinheit des RNA polymerase II

Die Transkription von proteincodierenden Genen wird in Eukaryonten durch RNA-Polymerase II und die Generellen Transkriptionsfaktoren, die an den Promotor eines Klasse II Genes binden, bewerkstelligt. Eine Regulation der Aktivitaet dieser molekularen Maschine erfolgt durch Aktivator- und Repressorproteine, die sequenzspezifisch an regulatorische Sequenzen dieser Gene binden. Fuer die Funktion von Aktivatoren ist nicht nur die Interaktion derselben mit der Transkriptionsmaschine sondern auch die Wechselwirkung mit akzessorischen Proteinen (Cofaktoren) essentiell. Diese fungieren als eine Art Transmitter fuer regulatorische Signale, welche den Zusammenbau bzw. die Aktivitaet der Maschine steuern. Die RNA-Polymerase II Transkription laesst sich in einem zellfreien in-vitro-System unter Verwendung einfacher Modellgene rekonstituieren. Dieses Testsystem wurde zur Identifizierung und Reinigung neuer Cofaktoren verwendet, von denen zwei in dieser Arbeit kloniert und naeher charakterisiert wurden. Einer dieser Faktoren (PAQ) ist Teil eines Multiproteinkomplexes, der Mediator genannt wird, und, wie in dieser Arbeit gezeigt, eine generelle regulatorische Funktion in der Zelle aufweist. Das zweite klonierte Protein (VACID) vermittelt spezifisch die Wirkung des Herpes simplex Virus Aktivators VP16, indem es sowohl an VP16 als auch an Mediator bindet und dessen Aktivitaet reguliert, was letztlich zu einer drastischen Stimulation der RNA-Polymerase II-Transkription fuehrt.

Biochemische Untersuchungen beschreiben NC2 als einen Transkriptionsrepressor, welcher stabil an das TATA-Box bindende Protein (TBP) bindet. Die spezifische Bindung von NC2 an TBP inhibiert die weitere Anlagerung der generellen Transkriptionsfaktoren TFIIA und TFIIB und führt dadurch zur Unterbrechung der Bildung des Initiationskomplexes. NC2 besteht aus zwei Untereinheiten, NC2a und NC2b, die starke Homologien zu den Histonen H2A bzw. H2B aufweisen. Alle Erkenntnisse zu Beginn dieser Arbeit basierten auf Beobachtungen, die in vitro erhalten wurden. Unklar sind die Funktionen von NC2 in der Zelle. Die Aufgabe dieser Arbeit bestand darin, ein in vivo-Modellsystem zu etablieren. Als Modellorganismus wurde die Bäckerhefe Saccharomyces cerevisiae ausgewählt. Hefe hat zwei Proteine, die stark homolog zum menschlichen NC2 sind. Beide sind essentiell für das vegetative Wachstum von Hefe. Für Plasmid-Austausch-Experimente wurden Hefestämme konstruiert, bei denen die chromosomalen Gene für NC2a und NC2b durch Wildtyp-Kopien auf einem URA3-Plasmid ersetzt wurden. Mit Hilfe einer negativen Selektion gegen das URA3-Gen in Anwesenheit von 5-FOA gelang es, die humanen NC2a- und NC2b-Gene als episomale Kopien in Hefe stabil einzubringen. So zeigte sich unter anderem, daß die beiden humanen NC2-Untereinheiten, sowohl einzeln in Kombination mit ihrem Dimerisierungspartner aus Hefe als auch gemeinsam in Form des menschlichen binären Komplexes, fähig waren, die physiologische Funktion ihres Gegenstückes aus Hefe zu übernehmen. Das gleiche System wurde auch eingesetzt, um Deletionsmutanten der humanen NC2-Gene in vivo zu untersuchen. Es wurde festgestellt, daß in beiden NC2-Untereinheiten die Domänen, welche für die in vivo-Funktion notwendig sind, die vom Mensch zur Hefe konservierten Regionen enthalten. Ein wesentlicher Teil dieser Arbeit bestand darin, spontane Suppressoren einer limitierenden NC2-Funktion in vivo zu isolieren und Suppressoren mit genomischen Punktmutationen zu charakterisieren. Gefunden wurde eine Punktmutation in der großen Untereinheit (Toa1) des Hefe-TFIIA, welche einen einzigen Aminosäure-Austausch von Valin zu Phenylalanin verursacht. Hefezellen, die diese Suppressor-Mutation in Toa1 (mt- Toa1) tragen, weisen einen Kälte-sensitiven Phänotyp auf und sind trotz fehlender NC2-Gene lebensfähig. Die biochemischen Eigenschaften des rekombinanten Proteins der Suppressor-Mutante wurden durch Gelretardations-, Footprinting- und in vitro Transkriptionsexperimente untersucht. Das Protein mt-Toa1 war in der Lage, stabile TFIIA-Komplexe zusammen mit der kleinen Untereinheit Toa2 auszubilden und die Rekrutierung von TBP an die TATA-Box auf dem Promotor zu unterstützen. Allerdings zeigten weitere Untersuchungen der Suppressor-Mutante, daß der ternäre Komplex aus mt-yTFIIA, TBP und DNA weniger stabil ist. Hinweise darauf gab die reduzierte Menge an Protein-DNA-Komplexen im Fall von mtyTFIIA in Gelretardationsexperimenten unter sättigenden Bedingungen. Das mt-yTFIIA verlor zugleich seine Antirepressionsaktivität in in vivo-Transkriptionsexperimenten in Anwesenheit von NC2. Die Isolierung und Charakterisierung der Suppressor-Mutante von NC2 lieferten zum ersten Mal den Beweis, daß die Genregulation in vivo eine präzise Balance zwischen positiv und negativ wirkenden Aktivitäten erfordert. Gleichzeitig bestätigen sie die in vitro Beobachtungen, insbesondere das Gleichgewicht zwischen TFIIA und NC2 in der Kompetition um das TATA-bindende Protein TBP. Eine weitere Aufgabe der Arbeit waren Mutagenese-Studien von humanem NC2 in vivo und in vitro. Innerhalb des Histone-Fold-Motives beider NC2-Untereinheiten wurden Punktmutanten isoliert, die ihre essentielle Funktion in der Hefezelle vollständig verloren haben. Es konnten Mutanten identifiziert werden, die Einfluß auf das Wachstum der Hefe mit dem Verlust der in vitro-Aktivität korrelierten. Die Charakterisierung dieser Mutanten lieferte erste Hinweise auf funktionelle Oberflächen von NC2, die für die ternäre Komplexbildung (NC2-TBP-DNA) und die Repressionsfunktion wichtig sind. Zusammengefaßt schafft die vorliegende Arbeit einen Einblick in die NC2-Funktion in der Zelle und erweitert unser Verständnis über den molekularen Mechanismus der Transkriptionsregulation während der Initiation der Klasse II-Transkription.