The Pennsylvania State University The Graduate School Department of Chemistry REGULATION OF THE PROTEIN KINASE PKR BY HIGHER-ORDER RNA SECONDARY AND TERTIARY STRUCTURES A Dissertation in Chemistry by Laurie A. Heinicke © 2010 Laurie A. Heinicke Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2010 ii The dissertation of Laurie A. Heinicke was reviewed and approved* by the following Philip C. Bevilacqua Professor of Chemistry Dissertation Advisor Chair of Committee Christine D. Keating Associate Professor of Chemistry Scott Showalter Assistant Professor of Chemistry Andrey Krasilnikov Assistant Professor of Biochemistry and Molecular Biology Barbara Garrison Professor of Chemistry Head of the Department of Chemistry *Signatures are on file in the Graduate School iii ABSTRACT A single strand of RNA can fold into multiple structures, often forming complex secondary and tertiary stuctures. Many factors influence RNA folding, including proteins, small molecules, salt, temperature, and pH. Human protein kinase PKR is a component of the innate immune response, and is activated by long stretches of double- stranded RNA (dsRNA), but is inhibited by highly structured or short dsRNAs. Activation of PKR often results in inhibition of protein synthesis. The types of RNAs encountered by PKR in the cell are diverse in structure and function, including mRNA, miRNA, tRNA, rRNA and viral RNAs. While PKR regulation by human cellular RNAs has not been extensively studied, it is known that PKR is activated by viral dsRNA genomes and long dsRNA replicative intermediates. The work presented in this thesis focused primarily on trapping and characterizing a unique RNA fold and then examining PKR regulation by that RNA. Experiments were performed using a diverse collection of viral and cellular RNAs, as well as model RNAs. Viral RNAs, including a small hairpin (TAR) from human immunodeficiency virus type 1 (HIV-I) and various length sequences from hepatitis delta virus (HDV), are single-stranded, but form complex secondary and/or tertiary structures. Both TAR RNA and the ribozyme portion of HDV are reported herein to form dimers and/or aggregates, which are found to be PKR activating species. Longer HDV sequences, which are rod-shaped and do not include continuous double-stranded RNA sequence greater than 20-bp, are still found to be potent PKR activators. Cellular RNAs have not been extensively examined as PKR regulators. It was our hyphothesis that most cellular RNAs have evolved defects to avoid activating or iv inhibiting PKR. We examined one cellular RNA, precursor miRNA 374a, which is reported herein to be an inhibitor of PKR. Precursor miRNAs are ~70-nt and are very structured with multiple bulges and mismatches. Removing defects and replacing them with Watson-Crick GC base pairs improves the ability of partially truncated pre-miRNAs to inhibit PKR. Another finding is that PKR activation at low pH is RNA-independent and represents a novel PKR response. Surprisingly, partially truncated pre-miRNA sequences were very potent inhibitors of RNA-independent activation of PKR at low pH, while only a weak inhibitors at high pH. Lastly, three separate studies were performed using model RNAs in an effort to understand the effects of RNA helical defects and dimerization on PKR activation, as well as to attempt to crystallize a minimal RNA binding register with p20 (dsRNA binding domain of PKR). The first study examined the effects of RNA helical defect position, size, and geometry on PKR activation. Positioning a bulge in the center of a helix, increasing bulge size, and orienting RNAs in cis or trans geometry decreased PKR activation relative to perfect double-stranded RNA. These results may be useful for predicting PKR activators. The second study investigated PKR activation by ternary complexes comprised of TAR dimers linked by base pairing to a bridging DNA oligo. Although all attempts to design a PKR activating complex were unsuccessful, rationale for designing complexes and native gel analyses are provided. Lastly, a third study involved preparing a ternary complex comprised of p20/stem-loop RNAs/U1A for crystallography. Preliminary gel-shift data and purification protocols are provided. Overall, the work in this thesis provides guidelines for predicting the ability of an RNA to activate or inhibit PKR. v TABLE OF CONTENTS LIST OF FIGURES ......................................................................................................... ix LIST OF TABLES .......................................................................................................... xii ABBREVIATIONS........................................................................................................xiii ACKNOWLEDGEMENTS ........................................................................................... xv Chapter 1 Introduction..................................................................................................... 1 1.1 RNA structure and folding..................................................................................... 1 1.2 PKR structure and function................................................................................... 4 1.3 Structure and function of PKR regulators ........................................................... 8 1.4 Methods used to analyze RNA structure and RNA-protein interactions ........ 10 1.5 Thesis objectives.................................................................................................... 12 Chapter 2 RNA Dimerization Promotes PKR Dimerization and Activation............ 15 2.1 Abstract.................................................................................................................. 15 2.2 Introduction........................................................................................................... 16 2.3 Materials and Methods......................................................................................... 19 2.3.1 Protein expression and purification ................................................................. 19 2.3.2 RNA preparation and purification ................................................................... 20 2.3.3 Native gel analysis ........................................................................................... 20 2.3.4 Purification of RNA from native gels.............................................................. 21 2.3.5 Mobility-shift assays........................................................................................ 22 2.3.6 PKR activation assays...................................................................................... 22 2.3.7 Enzymatic structure mapping of RNA............................................................. 23 2.3.8 Analytical ultracentrifugation .......................................................................... 24 2.4 Results .................................................................................................................... 24 2.4.1 Optimization of RNA monomer and dimer formation .................................... 24 2.4.2 Purification of RNA monomers and dimers from native gels ......................... 29 2.4.3 PKR activation assays using RNA monomers and dimers .............................. 31 2.4.4 Stoichiometry between p20 and RNA: Native gel analysis............................. 35 2.4.5 Affinity and Stoichiometry of PKR binding to TAR: AUC analysis .............. 38 2.4.6 Secondary structure of RNA monomers and dimers ....................................... 43 2.4.7 Dimerization and activation by scTAR variants.............................................. 48 2.5 Discussion............................................................................................................... 54 2.6 Acknowledgements ............................................................................................... 60 Appendix A Supplementary Figures for Chapter 2..................................................... 61 Chapter 3 Activation of PKR by the globular HDV ribozyme................................... 64 3.1 Abstract.................................................................................................................. 64 3.2 Introduction........................................................................................................... 64 3.3 Materials and Methods......................................................................................... 65 3.3.1 Protein expression and purification ................................................................. 65 3.3.2 Plasmid and RNA preparation ......................................................................... 67 vi 3.3.3 Native gel analysis ........................................................................................... 67 3.3.4 Purification of RNA from native gels.............................................................. 68 3.3.5 Mobility-shift assays........................................................................................ 69 3.3.6 PKR activation assays...................................................................................... 69 3.3.7 Enzymatic structure mapping of RNA............................................................. 70 3.4 Results and Discussion.......................................................................................... 71 3.4.1 PKR activation by heterogeneous 1/99 HDV variants .................................... 71 3.4.2 HDV aggregates activate