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MECHANISM of RNA REMODELING by DEAD-BOX HELICASES By MECHANISM OF RNA REMODELING BY DEAD-BOX HELICASES by QUANSHENG YANG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dr. Eckhard Jankowsky Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ___________________Quansheng Yang______________________________________ candidate for the__________________Ph.D._____________________degree (signed)__________William Merrick____________________________ (Chair of the Committee) ___________Vernon Anderson___________________________ ___________Eckhard Jankowsky _________________________ ___________Anthony Berdis ____________________________ ____________________________________________________ ____________________________________________________ (date) ___________12/12/2006______________________ 2 Table of Contents Chapter 1: Structures and biochemical activities of DExH/D helicases………………...11 Chapter 2: Initial characterization of Ded1…………….....…………………...................31 Chapter 3: ATP and ADP-dependent modulation of RNA unwinding and strand annealing activities by the DEAD-box helicase Ded1…………………………………...41 Chapter 4: Protein-assisted RNA structure conversion towards and against thermodynamic equilibrium values………………………………………………………68 Chapter 5: Duplex unwinding by a DEAD-box helicase without translocation on the loading strand...………………………………………………………………................101 Chapter 6: Duplex unwinding by DEAD-box helicases from both terminal and internal helical regions…………………………………………………………………………..125 Chapter 7: Future directions……………………………………………….……………146 Chapter 8: Materials and methods………………………………………………...…....151 3 List of Tables Table 1.1 NTPase activities of DExH/D helicases……………………………………....16 Table 1.2 Polarity of RNA duplex unwinding by DExH/D helicases…………………...18 Table 1.3 Unwinding capability of DExH/D helicases……………………………….....21 Table 1.4 A selection of DEAD-box helicases containing profound annealing activity...26 Table 2.1 Substrates and their sequences………………………………………………...34 Table 3.1 Substrates and their sequences………………………………………………...43 Table 4.1 Substrates and their sequences………………………………………………...72 Table 5.1 Substrates and their sequences used in Figure 5.1…………………………...105 Table 5.2 Sequences and characterization of substrates used in Figure 5.4 …………...110 Table 5.3 Sequences of multi-piece substrates (MPS)………………………………….114 4 List of Figures Figure 1.1 Structure and sequence characteristic of RNA helicases...…………………..14 Figure 2.1 Ded1 has RNA-dependent ATPase activity and ATP-dependent unwinding activity……………………………………………………………………………………33 Figure 2.2 Effects of the pH and salt concentration on the unwinding activity of Ded1 for 13 bp duplex RNA containing a 25 nt ssRNA at the 3’-end.......................……………...36 Figure 2.3 Effects of the pH and salt concentration on the unwinding activity of Ded1 for 13 bp blunt-end duplex ………………………….........……………………………...….37 Figure 3.1 RNA unwinding and strand annealing activities of Ded1…………….……...44 Figure 3.2 Pronounced strand annealing activity is specific for Ded1………….……….46 Figure 3.3 Ded1-catalyzed ATP-dependent steady state between RNA unwinding and strand annealing………………………………………………………………………….48 Figure 3.4 Dependence of unwinding and annealing rate constants on ATP-concentration and the nature of the RNA substrate……………………………………………………..50 Figure 3.5 ADP-dependent modulations of unwinding and annealing activities of Ded1...................................................................................................................................57 Figure 3.6 Effects of AMPPNP on unwinding and annealing activities of Ded1………..59 Figure 3.7 MgCl2-dependent modulation of unwinding and annealing activities of Ded1……………………………………………………………………………………...60 Figure 4.1 Substrate design and characterization………………………………………..71 Figure 4.2 Ded1 can unwind complex A and B and anneal complex A and B from respective RNA strands………………………………………………………………….74 Figure 4.3 RNA structure conversions…………………………………………………..75 Figure 4.4 Representative time course of structure conversion A Æ B with 800 nM Ded1 and 0.5 mM AMPPNP…………………………………………………………………...79 Figure 4.5 Basic kinetic scheme for structure conversion with Ded1 and ATP………....81 Figure 4.6 Ded1- assisted stabilization of tripartite intermediate………………………..83 Figure 4.7 Measurement of Ded1- assisted stabilization of tripartite intermediate by single molecule FRET……………………………………………………………………85 Figure 4.8 Basic kinetic schemes for Ded1-assisted structure conversion without ATP..89 Figure 4.9 Possible branch migration intermediates during Ded1-assisted structure conversion without ATP…………………………………………………………………90 Figure 4.10 Coupling of RNA structure conversion to deposition of U1A……………...93 Figure 4.11 Ded1-assisted RNA structure conversion…………………………………...95 Figure 5.1 Ded1 unwinds RNA duplexes irrespective of the orientation of single stranded regions…………………………………………………………………………………..104 5 Figure 5.2 Supplementing single stranded RNA in trans does not enhance the basal unwinding rate constant of the blunt-end substrates…………………………………....106 Figure 5.3 Equilibrium binding of different substrates by Ded1……………………….108 Figure 5.4 Unwinding of RNA-DNA hybrid substrates by Ded1……………………...109 Figure 5.5 Unwinding of multi-piece substrate I (MPS I) by Ded1 but not by NPH- II………………………………………………………………………………………..113 Figure 5.6 Unwinding of multi-piece substrate II by Ded1 but not by NPH-II………..116 Figure 5.7 Unwinding of the multi-piece substrate III by Ded1 but not by the DExH RNA helicase NPH-II…………………………………………………………..........……….118 Figure 5.8 Unwinding of MPS III components without streptavidin……….………….119 Figure 6.1 Dependence of unwinding rate constants on increasing Ded1 concentrations…………………………………………………………………….…….127 Figure 6.2 Unwinding of RNA-DNA chimeric substrates by Ded1…………………....129 Figure 6.3 Ded1 unwinds duplexes lacking any free RNA terminus…………………..131 Figure 6.4 Unwinding within the helical region by Ded1 and Mss116………………...133 Figure 6.5 Both Ded1 and Mss116 do not preferentially initiate unwinding from the terminus……………………………………………………………………………..…..137 Figure 6.6 The unwinding ability of both Ded1 and Mss116 decreases with the RNA length within the helical region………………………………………………………...140 Figure 6.7 DEAD-box helicases unwind RNA duplexes from both internal and terminal helical regions……………………………………………………................……….….142 6 Acknowledgements I would first like to thank my thesis committees, Drs. William Merrick, Vernon Anderson, and Anthony Berdis. I thank them for their very useful feedback on my project. I also thank Dr. William Merrick for giving me eIF4A and for reading the draft of this thesis. I thank Dr. Vernon Anderson for giving me RNA footing printing agent peroxynitrite and for his insight about enzyme kinetics. Many thanks to my advisor Dr. Eckhard Jankowsky. Without his continued support, it would have been impossible to finish this thesis. I am grateful for many insightful discussions with him, which were crucial to move the project forward. I also learned from him how to communicate scientific ideas effectively, including oral presentations and paper writing. Most importantly, the systematic way to investigate a problem I learned will continue to help me in my future career. I would like to thank Maggie Fairman for her initial introduction to the techniques for studying RNA helicases. I am in debt to her for letting me use her precious NPH-II preparation. I also thank her for explaining many crystal structures to me. I thank Heath Bowers for his initial studies on Ded1 and for his contribution to the idea of RNA structure conversion. His good ideas and insight about the helicase mechanism have been important to the progress of my project. I thank Dr. Nicholas Kay for helping me improve my proposal for PhD qualification and the introduction of this thesis. I want to thank Liu Fei for her technical support to plot the Figure 6.7 in this thesis. I thank Wen Wang for her optimization of the Ded1 purification and for helping construct Ded1 truncation mutants. I thank Dr. Mark Del Campo from Dr. Alan Lambowitz’s lab for producing purified Mss116. Finally, I would like to thank my parents and my wife for their unconditional love and support. I thank my daughter for highlighting my many days in Cleveland. 7 List of Abbreviations ADP adenosine diphosphate AMPPNP adenosine 5' (beta, gamma-imido) triphosphate ATP adenosine triphosphate bp base pair BSA bovine serum albumin Ded1 defines essential domain 1 DTT dithiothreitol EDTA (ethylenedinitrilo)-tetra acetic acid eIF4A eukaryotic initiation factor 4A EJC exon junction complex HEPES N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid MOPS 3-morpholinopropanesulfonic acid NP40 nonidet-P40 NPH-II nucleotide phosphohydrolase II nt nucleotide NTP nucleotide 5’-triphosphate PAGE polyacrylamide gel electrophoresis PEI polyethyleneimine RNP ribonucleoprotein SDS sodium dodecyl sulfate TLC thin layer chromatography TRAP the trp RNA-binding attenuation protein Tris tris(hydroxylmethyl)-aminomethane wt wild type 8 Mechanism of RNA Remodeling by DEAD-box Helicases Abstract by QUANSHENG YANG DEAD-box proteins remodel RNA duplexes and displace proteins from RNA in an ATP-dependent fashion. To understand how DEAD-box proteins remodel duplex RNA, I studied the mechanism of RNA remodeling by the DEAD-box protein Ded1 from S.cerevisiae. I found that Ded1 promotes
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