Erklärung H ierm it erkläre ich, d ass d ie vorliegend e Arbeit m it d em Titel " Insights into the RN A Polym erase II CTD cod e " von m ir selbstständ ig und ohne unerlaubte H ilfsm ittel angefertigt w ord en ist, und ich m ich d abei nur d er ausd rücklich bezeichneten Qu ellen und H ilfsm ittel bed ient habe. Die Dissertation ist w ed er in d er jetzigen noch in einer abgew and elten Form einer and eren Prüfungskom m ission vorgelegt w ord en. Ich erkläre w eiter, d ass ich m ich and erw

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... ig einer and eren Doktorprüfung ohne Erfolg nicht unterzogen habe. München, 27. Juni 2013 ________________ Roland Schüller Synopsis Synopsis Rpb1, the largest subunit of eukaryotic RNA Polymerase II (Pol II), contains a highly flexible structure at its C-terminus. This carboxyl-terminal domain (CTD) of Rpb1 is unique to eukaryotic organisms and consists of multiple tandemly repeated heptapeptides with the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 . Interestingly, the number of repeats differs from organism to organism and seems to correspond to genomic complexity, from 26 repeats in the yeast Saccharomyces cerevisiae to 52 repeats in the mammalian CTD (Chapman et al., 2008; Liu et al., 2010). Remarkably, five out of seven residues within the consensus sequence of the CTD can be potentially phosphorylated. In line with this, the production of monoclonal antibodies in our laboratory, against all different phosphosites within the heptad repeat confirmed the phosphorylation of Y 1 , S 2 , T 4 , S 5 and S 7 in vivo. Additionally to phosphorylation, other posttranslational modifications, such as cis-trans isomerisation of the two proline residues can also take place (Egloff et al., 2008) . The potential of the CTD to be modified at each residue can create a wide range of distinct combinations which could carry information that is essential at different steps of the transcription cycle, where the modifications can be recognized as a readable code, the so-called 'CTD code'. In this respect, the CTD might serve as a dynamic platform constantly signalling between the transcription machinery and factors that interact with Pol II (Buratowski et al., 2003; Corden et al., 2007). In this work, in order to gain new insights into the CTD code, CTD mutants were established to make the whole sequence accessible to mass spec ( MS) analysis and to map phosphosites within the CTD in vivo. MS results showed that the CTD can be phosphorylated within all 52 repeats revealing the existence of the full repertoire of possible phosphosites within the CTD in vivo. Moreover, individual CTD peptides displayed many different phosphorylation patterns reflecting the great diversity of phosphorylation signatures existing in parallel within the same CTD. Data produced in this thesis showed that mono-phosphorylated CTD repeats represent the prevailing phosphorylation form in vivo. Additionally, dominant phosphorylation signatures in di-phosphorylated (2P) CTD repeats could be mapped along the CTD by MS analysis. Tri-and tetra-phosphorylated (3P and 4P) CTD peptides were Synopsis detected as well, but only in very low amounts. By analysing 2P-signatures in more detail it was demonstrated that different 2P-combinations predominated within distinct repeats along the CTD, suggesting that CTD phosphorylation is location dependent. Finally, known CTD-protein binding motifs could be mapped and linked to specific CTD repeats. In conclusion, this work has established an approach for identifying high numbers of CTD-phosphosites, as well as high abundant CTD signatures along the whole CTD molecule, that contribute towards a better understanding of the 'CTD code' and open ways to yet undiscovered specific CTD-binding protein interactions. of the organism (Corden et al., 1985 and 1990; Chapman et al. 2008; Liu et al. 2010) (Figure 1). The ability of this repetitive sequence to interact with a wide range of nuclear factors is related to the dynamic plasticity of its structure and the diversity of binding surfaces generated by the multitude of posttranslational modifications it can accommodate. The association of specific posttranslational modifications of the CTD with particular events of the transcription cycle gave rise to the concept of the CTD code (Buratowksi et al.,