STRUCTURAL INSIGHTS INTO 7SK SNRNP COMPLEX AND ITS IMPLICATION FOR HIV-1 TRANSCRIPTIONAL CONTROL by LE LUO Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor Blanton S. Tolbert, Ph.D. Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January, 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Le Luo candidate for the degree of Doctor of Philosophy. Committee Chair Mary Barkley, Ph.D. Committee Member Paul Carey, Ph.D. Committee Member Fu-Sen Liang, Ph.D. Committee Member Blanton S. Tolbert, Ph.D. Date of Defense November 26, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein. Dedicated to my family and friends Table of Contents TITLE PAGE ........................................................................................................... COMMITTEE APPROVAL SHEET ......................................................................... DEDICATION .......................................................................................................... LIST OF FIGURES .............................................................................................. V ACKNOWLEDGEMENTS .................................................................................. VII LIST OF ABBREVIATIONS ................................................................................ IX ABSTRACT .......................................................................................................... 1 CHAPTER 1: INTRODUCTION ............................................................................ 3 1.1 History of AIDS/HIV discovery ............................................................... 4 1.2 HIV-1 Life Cycle ..................................................................................... 7 1.3 The HIV-1 structure and proteins ......................................................... 12 Major Structural Proteins ..................................................................... 13 Regulatory Proteins............................................................................. 15 Accessory Proteins ............................................................................. 17 I 1.4 The AIDS/HIV-1 Epidemics and Treatment ......................................... 21 1.5 HIV transcription by RNAPII ................................................................. 25 1.6 Transcriptional regulation of HIV-1 ...................................................... 29 1.7 7SK snRNPs in P-TEFb regulation ...................................................... 33 Reference .................................................................................................. 41 CHAPTER 2: DETERMINATION OF THE SECONDARY STRUCTURE OF 7SK SNRNA BY DMS-MAPSEQ ................................................................................ 53 2.1 Abstract................................................................................................ 54 2.2 Introduction .......................................................................................... 57 2.3 Materials and Method .......................................................................... 62 T7 RNA Polymerase preparation ........................................................ 62 7SK snRNA Preparation ..................................................................... 62 DMS modification ................................................................................ 63 RT-PCR .............................................................................................. 64 Next generation Sequencing ............................................................... 65 Processing of DMS-MaPseq data ....................................................... 65 Data validation and normalization ....................................................... 68 RNAStructure ...................................................................................... 69 2.4 Result................................................................................................... 70 II Population average model of DMS-MaPseq ....................................... 70 Preliminary Trial of Clustering on DMS_491 ....................................... 74 2.5 Discussion ........................................................................................... 76 Reference .................................................................................................. 89 CHAPTER 3: STRUCTURAL INSIGHTS INTO7SK SL3 AND ITS INTERACTION WITH HNRNP A1 ...................................................................... 93 3.1 Abstract................................................................................................ 94 3.2 Introduction .......................................................................................... 96 3.3 Materials and methods....................................................................... 103 RNA synthesis and purification ......................................................... 103 UP1 purification ................................................................................. 104 DMS-MaPseq .................................................................................... 104 Differential DMS-MaPseq .................................................................. 105 ITC .................................................................................................... 105 SAXS data Acquisition and analysis ................................................. 106 NMR data acquisition ........................................................................ 107 NMR titrations of UP1-7SK SL3up .................................................... 108 Structural modeling ........................................................................... 108 3.4 Results ............................................................................................... 110 III 7SK snRNA –hnRNP A1 (1:10) interaction ....................................... 110 Titration study of hnRNP A1- 7SK complexes ................................... 112 ITC experiment ................................................................................. 113 1H-1H NOESY and 1H-15N HSQC spectra of SL3S ........................... 114 SEC-SAXS model of SL3D and its complex ..................................... 115 7SK SL3D model fitted to SEC-SAXS model .................................... 116 Reference ................................................................................................ 133 CHAPTER 4: CONCLUSIONS AND FUTURE STUDIES ................................ 136 4.1 Conclusions ....................................................................................... 137 4.2 Future studies .................................................................................... 146 High resolution structural model of full length 7SK snRNA ............... 146 7SK associated snRNP study in vitro ................................................ 148 High resolution structure of 7SK SL3 - hnRNP A1 complex .............. 149 Reference ................................................................................................ 151 APPENDIX ....................................................................................................... 153 BIBLIOGRAPHY ............................................................................................... 155 IV List of Figures [Figure 1.1] The HIV Life Cycle. [Figure 1.2] Structure of HIV Virion. [Figure 1.3] Transcriptional Regulation of HIV Gene by P-TEFb. [Figure 1.4] Major 7SK snRNP Complexes Involved in P-TEFb Regulation. [Figure 2.1] Watson-Cricks Base Pairing of RNA. [Figure 2.2] Secondary Structure Model Determined Using Chemical and Enzymatic Probing by the J. Steitz Group. [Figure 2.3] Secondary Structure Model Determined Using SHAPE Method by the D. Price Group. [Figure 2.4] Secondary Structure Model Determined by DMS-MaPseq. [Figure 2.5] Two Preliminary Clustering Models Determined by DMS-MaPseq. [Figure 3.1] Structural Features of HnRNP A1. [Figure 3.2] HnRNP A1/ UP1 Titration into 7SK SL3up by EMSA. [Figure 3.3] Differential DMS-MaPseq of 7SK snRNA upon hnRNP A1 Binding. [Figure 3.4] Differential DMS Reactivity of 7SK-hnRNP A1 Complex in SL3D Region. [Figure 3.5] Secondary Structures of SL3 Constructs Used in the Study. [Figure 3.6] HnRNP A1 Titration into SL3up RNA Measured by ITC. [Figure 3.7] H2O NMR Spectrum of SL3S. [Figure 3.8] HnRNP A1 Titration to 7SK SL3up Measurement by 1H-15N HSQC. [Figure 3.9] SEC- SAXS Data of SL3D and Its 1:1 Complex with HnRNP A1. V [Figure 3.10] The Tertiary Structure Models for the 7SK SL3D Fitted into the Molecular Density Envelope Calculated from SEC-SAXS Data. VI ACKNOWLEDGEMENTS I would like to begin by thanking my Ph.D. advisor, Dr. Blanton S. Tolbert for his guidance during my Ph.D. career since Miami University. He is a creative and talented scientist who is always encouraging to try out and learn new techniques, which allowed for the work presented in this thesis to be possible. More importantly, his supports made it possible for me to always learn and practice state of the art techniques and learn from great scientists that transformed me from a student to a researcher. To my dearest new family, the Tolbert group, we are from everywhere and we all have been this great family, contributing to it and getting nurtured by each other. I sincerely appreciated the time we spent with each other; all