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RNA-Protein Interactions of a Kink

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RNA-Protein Interactions of a Kink Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2005 Biophysical and Biochemical Investigation of an Archaeal Box C/D SRNP: RNA- Protein Interactions of a Kink Turn RNA within the Functional Enzyme Terrie Luong Moore Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES BIOPHYSICAL AND BIOCHEMICAL INVESTIGATION OF AN ARCHAEAL BOX C/D SRNP: RNA-PROTEIN INTERACTIONS OF A KINK TURN RNA WITHIN THE FUNCTIONAL ENZYME By TERRIE LUONG MOORE A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Summer Semester, 2005 The members of the Committee approve the dissertation of Terrie Luong Moore defended on May 3, 2005. ________________________________ Hong Li Professor Directing Dissertation ________________________________ Lloyd M. Epstein Outside Committee Member ________________________________ Timothy M. Logan Committee Member ________________________________ John G. Dorsey Committee Member Approved: ___________________________________________________ Naresh Dalal, Chair, Department of Chemistry and Biochemistry The Office of Graduate Studies has verified and approved the above named committee members. ii I dedicate this work to my husband, Michael, for being there every step of the way and always giving me hope about the future. Without your love and support, this work would not have been possible. iii ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Hong Li, first and foremost for taking me into her lab and allowing me to grow intellectually. I would like to thank all of my committee members, Dr. Timothy Logan, Dr. Lloyd Epstein, and Dr. John Dorsey, for their knowledge and guidance during my graduate career. I am very thankful for my husband, Michael Moore, who believed in me even when I didn’t and kept me going even when the waters were rough. I want to thank all my family for their love and support. They have always told me that I could do whatever I wanted to do. I want to thank all the members of the Li lab especially Rumana Rashid and Sri Oruganti for all the coffee and laughs and for keeping me sane. Thanks to Laura Jane Phelps, Meredith Newby Lambert, and Kersten Schroeder for helping me through the rough times. I would like to thank Soma for all his help with crystal diffraction and the multiple discussions on many other topics in the lunch room. Thank-you to Dr. Jack J. Skalicky for his NMR help and career advice. I would like to thank Marcia O. Fenley for her help on the electrostatics work. I would like to thank Daphne Williams and her little man for making me smile even when I didn’t want to. I would also like to thank (Kitty) Kat Phipps for sharing my goofy sense of humor. And last, but definitely not last, thanks to Christina Wimberly, my best friend, for helping me relax when I needed it the most and understanding things that nongraduate people would normally not understand. My time here has taught me so much about science and about life. I will miss all the friends I made here and I will miss Tallahassee a lot more than I ever imagined. Good luck in your future endeavors and may our paths cross again. I would also like to acknowledge the funding from the NIH and AHA agencies for the box C/D project. iv TABLE OF CONTENTS List of Tables vii List of Figures viii List of Abbreviations x Abstract xii INTRODUCTION 1 Noncoding RNAs 1 Small Nucleolar Ribonucleoprotein Particles 2 History of snRNAS 6 Archaeal Homologs of Small Nucleolar RNAs 8 Location of SnoRNA genes 9 Significance of Nucleotide Modifications 10 Box C/D SnoRNPs 13 CRYSTAL STRUCTURE AND MOLECULAR DETAILS OF A BOX C/D RNA-L7AE COMPLEX 19 Introduction 19 Results and Discussion 23 Conclusion 52 Materials and Methods 54 BOX C’/D’ RNA-L7AE CRYSTALLIZATION AND PRELIMINARY X-RAY DIFFRACTION DATA 58 Introduction 58 Results and Discussion 59 Conclusion 64 Materials and Methods 66 DESIGN AND CRYSTALLIZATION OF AN ENTIRE BOX C/D SNORNP 68 Introduction 68 Results and Discussion 68 Conclusion 78 Materials and Methods 78 CONCLUSIONS 80 REFERENCES 82 v BIOGRAPHICAL SKETCH 90 vi LIST OF TABLES 1. Data Collection, Phasing and Refinement Statistics 25 2. Crystallization Trials for L7Ae-box C'/D' RNA Co-crystal 61 3. Optimization of L7Ae-Box C'/D' 26mer Crystal 62 4. Crystallization Trials for Box C/D sRNP Supercomplex 75 5. Optimization of Supercomplex Crystal with Constructs S4, S5, and S6 76 vii LIST OF FIGURES 1. Ribonucleoprotein Particles 3 2. Classes of snoRNAs 5 3. U3 snoRNA Secondary Structure 7 4. Modifications in the E. Coli and yeast rRNA 11 5. RNA Sugar Conformers 12 6. Models of Box C/D snoRNP and sRNP Assembly 15 7. Crystal Structure of AF Fibrillarin-Nop5p Complex 16 8. Representations of Protein-Kink turn Complexes 21 9. Kink Turn RNA Sequences 22 10. RNA Constructs for Structural Characterization with L7Ae 26 11. EMSA of Box C/D RNA Constructs with L7Ae 27 12. L7Ae-box C/D RNA Crystal 27 13. Electron Density Map of L7Ae-box C/D RNA Complex 28 14. Crystal Arrangement of L7Ae-box C/D RNA 25mer Crystal 30 15. L7Ae Structure and Alignment 31 16. Surface Electrostatic Potentials of AF L7Ae, human 15.5 kD, and HM L7Ae 33 17. Structure of Box C/D 25mer RNA 34 18. Structural Features of Box C/D RNA Kink Turn 36 19. Structural Comparison of Three K-turn RNAs Bound with the 15.5 kD/L7Ae protein 38 20. Comparison of Electrostatic Surface Potentials of Three Kink Turn RNAs 40 21. Comparison of K-turn Orientations with Respect to Their Bound Proteins 41 22. Superimposed Complexes of 15.5kD-U4 snRNA and L7Ae-box C/D RNA 43 23. Overall Crystal Structure of the L7Ae-box C/D RNA Complex 44 24. Comparison of L7Ae-box C/D RNA Contacts with the 15.5 kD-U4 5‘-stem RNA and HM L7Ae-KT-15 Complexes 46 25. Base Pairing that Gives Rise to Imino Resonances 48 26. 1D spectra of L7Ae in the Absence and Presence of Box C/D RNA 50 viii 27. 1D NMR Spectra of Box C/D 21mer RNA Titration with 15N-L7Ae 51 28. Box C’/D’ RNA Constructs for Crystallization with L7Ae 60 29. EMSA of Box C’/D’ Constructs with L7Ae 60 30. Pictures of L7Ae-box C’/D’ 26mer RNA Crystals 63 31. 1D Spectra of box C’/D’ 20mer RNA with L7Ae 65 32. Native Constructs for Supercomplex Crystallization 69 33. Oligonucleotide Box C/D RNA Constructs 71 34. EMSA of Supercomplex RNA Constructs with L7Ae and Fibrillarin- Nop5p Complex 73 35. Box C/D sRNP Crystal with Construct S4 77 ix LIST OF ABBREVIATIONS AF: Archaeoglobus fulgidus A: adenosine ATP: adenosine triphosphate C: Cytidine CS: canonical stem CNS: Crystallography NMR System DNA: Deoxyribonucleic acid EDTA: Ethylene diamine tetra acetate EM: Electron microscopy EMSA: electrophoretic mobility shift assay FPLC: fast performance liquid chromatography G: guanosine monophosphate HM: Haloarcula marismortue HPLC: High-performance liquid chromatography Kt or K-turn: kink turn MAD: multiple wavelength anomalous diffraction miRNA: micro RNA MJ: Methanococcus jannaschii mRNA: messenger RNA MRP: mitochondrial RNA processing NAIM: nucleotide analog interference modification ncRNA: noncoding RNA NCS: noncannonical stem PBE: Poisson-Boltzmann equation PEI: polyethyleneimine PEG: polyethylene gylcol R-M enzyme: restriction modification enzyme x RNA: ribonucleic acid RNase: ribonuclease RNP: ribonucleoprotein particle rRNA: ribosomal RNA SAM: S-adenosyl-l-methionine SBP: SECIS binding protein SECIS: selenocysteine insertion sequence scRNPs: small cytoplasmic RNPs siRNAs: small interfering RNAs snoRNA: small nucleolar RNA snoRNPs: small nucleolar RNP snRNA: small nuclear RNA snRNP: small nuclear RNP sRNA: small RNA sRNP: small RNP SRP: signal recognition particle TBE: tris-Borate EDTA Tt: Thermus thermophilus U: uridine UTP: uridine triphosphate UV: ultraviolet electromagnetic radiation WC: Watson-Crick base pair xi ABSTRACT Box C/D snoRNPs catylze the specific 2’O-methylation of rRNA in important regions the ribosome, although the role of the modifications is unclear. Eukaryotic box C/D snoRNPs consists of a box C/D RNA and four proteins, Fibrillarin, Nop56, Nop58, and 15.5kD. Archaeal homologs are simiplified containing three proteins, L7Ae, Nop5p, and Fibrillarin with a box C/D RNA. The box C/D sequences are proposed to form the recently recognized kink turn structure which is found in many types of RNA. Most are associated with proteins and protein binding may nucleate the assembly of other proteins onto the RNA. Dissecting the structure and biochemical properties of box C/D snoRNPs may not only help in understanding the function of the modifications, but may also give insight into the role of kink turn RNAs in RNP assembly. An archaeal box C/D RNA embedded within the intron of pre-tRNATrp from Archaeglobus Fulgidus(AF) that guides two modifications in the tRNA was used as the model for the investigation of three complexes: L7Ae-box C/D RNA, L7Ae-box C’/D’ RNA, and the entire box C/D sRNP. Extensive crystallization trials resulted in crystals for each complex. A crystal structure of the box C/D RNA-L7Ae complex was determined to 2.7Å and shows the box C/D sequences do form a kink turn. Detailed structural comparisons of the AF L7Ae-box C/D RNA complex with previously determined crystal structures of L7Ae homologs in complex with functionally distinct kink turn RNAs revealed a conserved RNA-protein interface suggesting a conformational “adaptability” of the kink turn RNAs in binding L7Ae homologs.
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