Functional architecture of RNA polymerase I.

Abstract

Synthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. Here we report the 12 A cryo-electron microscopic structure for the complete 14-subunit yeast Pol I, a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3'-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 3'-end trimming.

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Dissertation zur Erlangung des Doktorgrades der Fakultät für
Chemie und Pharmazie der Ludwig-Maximilians-Universität München



Functional Architecture
of RNA Polymerase I





Claus-Dieter Kuhn
aus Mutlangen

2008

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Erklärung

Diese Dissertation wurde im Sinne von §13 Abs. 3 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Patrick Cramer betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, den 8. Januar 2008












Dissertation eingereicht am 9. Januar 2008
1. Gutachter: Prof. Dr. Patrick Cramer
2. Gutachter: Prof. Dr. Roland Beckmann
Mündliche Prüfung am 27. Februar 2008

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Acknowledgements
Summary
Part I: Introduction
I.1 | Eukaryotic RNA polymerases...................................................................1
I.2 | General importance of rDNA transcription................................................2
I.3 | Structural organization of rDNA loci..........................................................3
I.4 | Epigenetics...............................................................................................5
I.5 | The RNA polymerase I transcription cycle................................................7
I.6 | In vivo regulation of rDNA transcription ....................................................9
I.7 | Making ribosomes...................................................................................10
I.8 | Structural studies on eukaryotic RNA polymerases................................12
I.9 | Aim of this study .....................................................................................13
Part II: Results and Discussion
II.1 | Large-scale purification of RNA polymerase I........................................14
II.2 | Crystallization of Pol I............................................................................18
II.3 | Cryo-crystallography and heavy atom derivatization .............................21
II.4 | Data collection and processing..............................................................23
II.5 | Attempts on solving Pol I by X-ray crystallography................................26
II.6 | Cryo-EM structure of Pol I at 12 Å resolution.........................................31
II.7 | Homology model of the Pol I core explains EM density.........................35
II.8 | Crystal structure of A14/43 elucidates Pol I initiation.............................38
II.9 | A49 and A34.5 act as built-in, heterodimeric elongation factor..............41

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II.10 | Pol I has intrinsic RNA cleavage activity that requires A12.2...............47
II.11 | Conclusions.........................................................................................51
Part III: Materials and Methods
III.1 | Purification of RNA Polymerase I from S. cerevisiae............................53
III.2 | Purification of Pol I variants..................................................................56
III.3 | Crystallization of Pol I...........................................................................57
III.3.1 | Crystallization by vapor diffusion....................................................57
III.3.2 | Streak-seeding...............................................................................58
III.3.3 | Crystal harvesting and cryo-protection...........................................58
III.3.4 | Heavy atom derivatization and crystal freezing..............................59
III.4 | Data collection......................................................................................59
III.5 | Data processing....................................................................................60
III.6 | Attempts on structure solution ..............................................................61
III.6.1 | Experimental phasing.....................................................................61
III.6.2 | Molecular replacement...................................................................61
III.7 | Cryo-electron microscopy of Pol I.........................................................63
III.7.1 | Negative stain.................................................................................63
III.7.2 | Preparation of grids........................................................................63
III.7.3 | Cryo-EM data collection.................................................................64
III.7.4 | Image processing for 14-subunit Pol I............................................64
III.8 | Cryo-EM data processing for 12-subunit Pol I ΔA49/34.5.....................66
III.9 | Modeling of the Pol I core.....................................................................67

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III.10 | Structure prediction of A49/34.5 .........................................................67
III.11 | Purification of recombinant A49/34.5..................................................68
III.12 | Yeast genetics....................................................................................69
III.12.1 | 6-azauracil phenotyping of GPY2 ΔRPA34..................................69
III.12.2 | Cloning and fermentation of GPY2 RPA12ΔC..............................70
III.13 | In vitro RNA assays............................................................................70
III.13.1 | RNA extension assays using a minimal scaffold..........................70
III.13.2 | RNA extension assays using a complementary bubble................71
III.13.3 | RNA cleavage assays.................................................................72
III.13.4 | Electrophoretic mobility shift assay (EMSA)................................72
III.14 | Figure preparation ..............................................................................72
Part IV: Appendix
IV.1 | DENZO and SCALEPACK scripts........................................................73
IV.2 | Self-rotation function scripts.................................................................76
IV.3 | SOLVE scripts......................................................................................78
IV.4 | MOLREP script.....................................................................................81
IV.5 | Initial cryo-EM processing for 14-subunit Pol I .....................................82
IV.6 | Initial cryo-EM processing for Pol I ΔA49/34.5 .....................................84
IV.7 | Sequence alignments...........................................................................85
Part V: References..........................................................................................91
Curriculum vitae