University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 6-17-2008 Cleavage and Ligation Studies in Hairpin and Hammerhead Ribozymes Using Site Specific Nucleotide Modifications Snigdha Roy University of Vermont Follow this and additional works at: http://scholarworks.uvm.edu/graddis Recommended Citation Roy, Snigdha, "Cleavage and Ligation Studies in Hairpin and Hammerhead Ribozymes Using Site Specific ucleN otide Modifications" (2008). Graduate College Dissertations and Theses. Paper 203. This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. CLEAVAGE AND LIGATION STUDIES IN HAIRPIN AND HAMMERHEAD RIBOZYMES USING SITE SPECIFIC NUCLEOTIDE MODIFICATIONS A Dissertation Presented by Snigdha Roy to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Microbiology and Molecular Genetics February, 2008 ABSTRACT RNA catalysis is of fundamental importance in many biological functions, such as the peptidyl transferase activity of the ribosome and genetic control by riboswitches, among others. Small ribozymes are a convenient system to increase our understanding about the structure, folding and catalytic mechanism of ribozymes. This dissertation includes analysis of certain aspects of the catalytic mechanism in the hairpin and hammerhead ribozyme. In the hairpin ribozyme, we studied the functional consequences of molecular substitutions at two conserved positions, A9 and A10. These nucleotides are located close to the scissile phosphate but their exact function is unclear since they do not appear to be making any essential interactions with other nucleotides in the catalytic core. G, C, U, 2-aminopurine, 2, 6-diaminopurine, purine, and inosine were substituted at A9 and A10 and their effects on cleavage and ligation rates were analyzed. The effect of the variations on tertiary structure and docking was monitored by hydroxyl radical footprinting and native gel electrophoresis. It was observed that all the variants that exhibited poor docking and/or tertiary structure formation were also ligation challenged whereas they performed normally in the cleavage reaction. We found a unique variant, A10G that cleaved five times faster than A10 but did not exhibit any ligation. Results suggested that ligation required a more kinetically stable core than that needed to carry out cleavage. The hammerhead ribozyme field featured extensive disagreements between the crystal structure of the minimal hammerhead released in the mid 90s and the accumulating biochemical data. Much of the conflict was resolved with the new crystal structure of the extended hammerhead ribozyme. This structure confirmed many of the biochemical findings and brought out some new interactions, notably the G8·C3 base pair. We studied numerous base substitutions to establish the importance of the base pair for cleavage and ligation. Catalysis requires the formation of the base pair but even the fastest base paired variant was 10-fold slower than G8·C3 base pair. Docking and tertiary structure analysis by hydroxyl radical footprinting and native gel electrophoresis emphasized the importance of having a purine at position 8 and a pyrimidine at 3. Catalysis in the unmodified ribozyme was uniquely accompanied by hydrolysis of the 2 ′, 3 ′- cyclic phosphate ring present on one of the cleavage products, leading to the generation of non-ligatable products during a ligation assay. We determined the ligation rate-pH profile for unmodified ribozyme that differed from the cleavage rate-pH profile only at high pH. CITATION Material from this dissertation has been published in the following form: Gaur, S., Heckman, J. E. and Burke, J. M. (2008) Mutational inhibition of ligation in the hairpin ribozyme: Substitutions of conserved nucleobases A9 and A10 destabilize tertiary structure and selectively promote cleavage. RNA 14(1), 55-65 ii ACKNOWLEDGEMENTS A thesis is never complete without acknowledging the people who have contributed in different ways to help me complete my thesis. Firstly I would like to thank Dr. John Burke, I could not have asked for a better advisor and a mentor for my Ph.D research. He has been very encouraging and exposed me to better learning opportunities and has also been very patient and understanding when my experiments did not give the desired results. I am happy and grateful to him that I could be a part of his lab as this has been an incredible learning experience. I would also like to thank Dr. Joyce Heckman who made my transition into the lab very smooth. I am grateful for the time and patience she took to teach me most of the techniques and more importantly the rationale behind each of them. Over the years, I have learnt a lot more than just science from her. She was always there whenever I needed help, moral or science related. I would like to thank my committee members for providing me helpful suggestions and keeping me on track towards completing my Ph.D. Anne Macleod has been very helpful and a really good friend. I would also like to thank Dr. Ken Hampel and Dr. Iwona Buskiewicz for helpful discussions and also teaching me to question and critically analyze experimental data. It was a great learning experience to work with Dr. Dominic Lambert (previously a graduate student in our lab) who taught me the concept of multi- tasking and was a role model to me. A special thanks goes to all my friends here, iii especially Amalthiya and Karunya who have been a great support to me while I was far away from my family and friends. I can never thank my parents and family enough for their support and understanding during the whole time that I was here. Last but not the least I would like to thank my husband who has been immensely patient and encouraging while I completed my thesis. iv TABLE OF CONTENTS CITATION.......................................................................................................................ii ACKNOWLEDGEMENTS ............................................................................................iii LIST OF TABLES ........................................................................................................viii LIST OF FIGURES.........................................................................................................ix CHAPTER 1.....................................................................................................................1 Introduction ......................................................................................................................1 RIBONUCLEASE A ....................................................................................................2 HAIRPIN RIBOZYME ................................................................................................5 Origin ..........................................................................................................................5 Secondary structure.....................................................................................................5 Tertiary Structure ........................................................................................................9 Role of metal ions .....................................................................................................12 Catalytic Mechanism.................................................................................................13 HAMMERHEAD RIBOZYME..................................................................................16 Origin ........................................................................................................................16 Secondary Structure ..................................................................................................17 Folding ......................................................................................................................19 Tertiary Structure and functional correlation............................................................ 21 Catalytic Mechanism.................................................................................................23 CHAPTER 2...................................................................................................................51 v Mutational Inhibition of ligation in the hairpin ribozyme: Substitutions of conserved nucleobases A9 and A10 destabilize tertiary structure and selectively promote cleavage ........................................................................................................................................51 ABSTRACT................................................................................................................52 INTRODUCTION ......................................................................................................53 MATERIALS AND METHODS................................................................................57 RESULTS ...................................................................................................................61 DISCUSSION .............................................................................................................69 ACKNOWLEDGEMENTS.......................................................................................