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Reconstitution of a Polii-Like RNA Polymerase and Contribution of Subunit E’ and Structural Elements in the Active Center to RNA Polymerase Functions

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Reconstitution of a Polii-Like RNA Polymerase and Contribution of Subunit E’ and Structural Elements in the Active Center to RNA Polymerase Functions Reconstitution of a PolII-like RNA Polymerase and Contribution of Subunit E’ and Structural Elements in the Active Center to RNA Polymerase Functions Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät III – Biologie und Vorklinische Medizin der Universität Regensburg vorgelegt von Souad Naji aus Rabat, Marokko Regensburg, im August 2006 Promotionsgesuch eingereicht am: 18.07.2006 Die Arbeit wurde angeleitet von: Prof. Dr. M. Thomm Pruefungsausschuss: Vorsitzender: Prof. Dr. R. Wirth 1. Gutachter und Pruefer: Prof. Dr. M. Thomm 2. Gutachter und Pruefer: Prof. Dr. H. Tschochner 3. Pruefer Prof. Dr. R. Sterner Table of contents 1 Table of contents Table of contents .........................................................1 I Introduction .................................................................5 1. The transcription cycle......................................................................................... 5 2. DNA-dependent RNA polymerase....................................................................... 6 2.1 Bacterial RNAP ...................................................................................................... 7 2.2 Eukaryotic RNAPs.................................................................................................. 7 2.3 Archaeal RNAP .................................................................................................... 10 3. General RNAP architecture............................................................................... 11 4. RNAPII structure................................................................................................ 13 4.1 The clamp ............................................................................................................. 15 4.2 The active site ....................................................................................................... 16 4.3 RNAPII elongation complex structure ................................................................. 17 5. Purpose of this work ........................................................................................... 19 II Material.......................................................................20 1. Suppliers .............................................................................................................. 20 1.1 Chemicals.............................................................................................................. 20 1.2 Enzymes, antibodies and others proteins.............................................................. 21 1.3 Kits ....................................................................................................................... 21 1.4 Chromatography equipment and columns ............................................................ 22 2. Materials for cloning........................................................................................... 22 2.1 Archaeal strain ...................................................................................................... 22 2.2 Bacterial strains..................................................................................................... 22 2.3 Plasmids................................................................................................................ 22 2.4 Oligonucleotides ................................................................................................... 23 III Methods .....................................................................25 1. Purification of endogenous Pfu RNAP.............................................................. 25 Table of contents 2 2. Preparation of competent bacterial cells .......................................................... 26 3. Cloning strategies................................................................................................ 26 3.1 DNA digestions and ligations............................................................................... 27 3.2 Construction of B and A’ mutants with site directed mutagenesis....................... 27 3.3 Bacterial Transformation ...................................................................................... 28 3.4 Screening of transformants ................................................................................... 28 4. Overexpression of recombinant subunits ......................................................... 29 5. Recombinant subunit purification .................................................................... 29 5.1 Purification of insoluble subunits ......................................................................... 29 5.2 Purification of soluble subunits ............................................................................ 30 6. In vitro reconstitution of recombinant RNAP and RNAP sub-complexes..... 31 7. Promoter-Independent in vitro Transcription Assays..................................... 31 8. Promoter-Directed in vitro Transcription Assays............................................ 32 9. Electrophoretic Mobility Shift Assays (EMSAs).............................................. 33 10. Western-blotting and immunodetection of proteins........................................ 33 11. Pfu RNAP auto-phosphorylation....................................................................... 34 IV Results .......................................................................35 1. Purification of endogenous Pfu RNAP and its recombinant subunits........... 35 2. Is E” a subunit of Pfu RNAP? ........................................................................... 37 3. In vitro assembly of Pfu RNAP subunits........................................................... 38 4. Recombinant RNAP activity in promoter-directed in vitro transcription..... 38 5. The importance of individual small subunits of Pfu RNAP............................ 40 6. Functions of F and E’ subunits of Pfu RNAP................................................... 43 6.1 In vitro transcription of RNAP∆E’F..................................................................... 43 6.2 Interactions between E’, F and TFE ..................................................................... 43 6.3 Interactions between E’, F and E” ........................................................................ 45 6.4 Effects of E’ and F on in vitro transcription at low temperatures......................... 45 6.5 Effect of E’ on promoter opening during transcription initiation......................... 47 7. The BDLNP sub-complex associates with promoter bound TBP-TFB.......... 48 7.1 In vitro assembly of Pfu RNAP sub-complex BDLNP ........................................ 48 Table of contents 3 7.2 Electrophoretic mobility shift assay of BDLNP with promoter-TBP-TFB complex ...................................................................................................................... 48 8. Functional investigation of Pfu RNAP lid, rudder, fork 1 and fork 2 ........... 50 8.1 Promoter-independent in vitro transcription assays of RNAP mutants................ 52 8.2 Promoter-directed in vitro transcription assays of RNAP mutants....................... 52 8.3 At which steps of the transcription pathway are lid, rudder and fork2 important for the activity? ............................................................................................................. 53 9. Autophosphorylation of Pfu RNAP................................................................... 57 10. Function of Pfu RNAP phosphorylation in the transcriptional activity........ 57 V Discussion.................................................................59 1. Purification of endogenous Pfu RNAP.............................................................. 59 1.1 Transmission electron microscopy of Pfu RNAP................................................. 59 1.2 E” is not a subunit of Pfu RNAP .......................................................................... 60 2. Reconstitution of active recombinant Pfu RNAP ............................................ 60 3. The role of individual subunits in the catalytic activity of RNAP.................. 61 3.1 Subunit K .............................................................................................................. 61 3.2 Subunit H .............................................................................................................. 62 3.3 Subunits P and N................................................................................................... 62 3.4 Subunits E’ and F.................................................................................................. 63 4. E’ stimulates transcription at lower temperatures.......................................... 66 5. E’ catalyzes open complex formation during transcription initiation ........... 66 6. F interacts with the transcription factor TFE.................................................. 68 7. The BDLNP sub-complex associates with promoter bound TBP-TFB.......... 69 8. Function of RNAP loop structural elements .................................................... 70 9. Autophosphorylation of Pfu RNAP................................................................... 73 VI Summary....................................................................74 VII References.................................................................76 VIII Appendix .....................................................................86 1. List of abbreviations ..........................................................................................
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