ABSTRACT CANTARA, WILLIAM ANTHONY. Arginyl-tRNA Modifications Modulate Anticodon Domain Structure, Function and Dynamics in Escherichia coli. (Under the direction of Paul F. Agris) During ribosome-mediated protein synthesis, non-initiator tRNAs act as translating chaperones by transferring the correct amino acid to the ribosome through sequence-specific interactions with mRNA codons in the ribosomal A-site. To achieve uniformity in codon recognition ability between different tRNAs, the anticodon domains of all must adopt a singular conformation in the ribosomal decoding center. Since there is a necessary sequence variation for the three anticodon residues, innovative ways of attaining a constant loop structure are required. Nature has resolved this issue by introducing functional groups to the loop residues, changing the chemical environment and, thus, the conformation. In fact, there is a large diversity and number of these modifications found in tRNAs of all known life. Escherichia coli (E.coli) arginyl-tRNAs offer the opportunity to study these modifications in the context of six-fold degenerate codons that vary widely in their cellular usage. A single Arg1 Arg2 set of very similar isoacceptors, tRNA ICG and tRNA ICG, have anticodon domains that 2 differ only in the presence or absence of a very rare modification, 2-thiocytidine (s C32). 2 These tRNAs are particularly interesting not only because of the rare s C32 modification, but also through the wobble position modification I34, they are both responsible for decoding two very common codons CGU and CGC as well as the very rare CGA codon. Typically, the codon usage correlates with the tRNA content of the cell; however, in this instance, the tRNA content is high which does not match what would be expected for an isoacceptor that recognizes a rare codon. Here we have shown conclusively that modifications can act to restrict the decoding capability of the tRNA to only the two common codons, suggesting that the tRNA is present at high levels, consistent with decoding common codons, but that there is a smaller pool of tRNA in different states of modification, corresponding to decoding of the rare codon. The mechanism of this function is not completely understood, however, we show that the modifications do not accomplish this through a completely structural device. In this case, modifications act to modulate the flexibility of specific loop residues, thereby modulating the conformational landscape that must be traversed between two disparate, but necessary, cellular conformations. It is clear from many previous studies that modifications can perform specific functions in the anticodon stem and loop (ASL), most notably at position 34 which is known to facilitate Arg4 the “wobble” recognition of multiple codons. E.coli tRNA UCU offers a novel function of modification at this position that is not fully understood. In a very similar isoacceptor, Lys partially modified tRNA UUU, a very different decoding function is seen. Both of these Arg4 tRNAs contain the same loop sequence with the exception that tRNA UCU has a C at Lys position 35, whereas tRNA UUU has U35. Interestingly, they both contain the same loop 5 6 Arg4 modifications, mnm U34 and t A37, with the exception that tRNA UCU has an additional 2 Lys s C32 modification; however tRNA UUU, in this particular modification state can decode Arg4 2 both AAA and AAG and tRNA UCU decodes only AGA. Although s C32 enhances codon Arg1 discrimination in tRNA ICG, codon-specific ribosome binding assays clearly refute this role Arg4 in tRNA UCU. The modifications, however, appear to impart similar biophysical and Lys thermodynamic properties to the ASL as in tRNA UUU, therefore a full structural characterization is underway using NMR spectroscopy to ascertain whether modifications Lys drive the conformation toward a canonical U-turn conformation as with tRNA UUU. Preliminary evidence indicates a role for modifications in altering the stability of the non- canonical C32•A38 base pair through stable stacking interactions and elevation of A38H1 pKa. Arginyl-tRNA Modifications Modulate Anticodon Domain Structure, Function and Dynamics in Escherichia coli by William Anthony Cantara A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Biochemistry Raleigh, North Carolina 2012 APPROVED BY: _______________________________ ______________________________ Paul Wollenzien Paul F. Agris Committee Chair _______________________________ ______________________________ E. Stuart Maxwell James W. Brown DEDICATION To Shraddha, my loving wife, who keeps me on the right path through life and is someone that I can always count on. Also, to my mother and other family members for their sacrifices throughout my life and for nurturing my curiosity. Finally, to my friends and labmates for their support and advice. ii BIOGRAPHY William Anthony Cantara spent part of his childhood in York, Pennsylvania until moving to Grassflat, Pennsylvania during kindergarten. He spent his childhood and high school years enjoying rural life and participating in athletics. He attended Juniata College, where he met his wife and graduated with a bachelor’s degree in Biochemistry and Physics. During college, his lab experiences with Dr. Jill Keeney showed him that research was his true calling. He then moved to Raleigh, NC with his future wife to work under the guidance of Dr. Paul Agris at North Carolina State University. Will looks forward to using the skills and experiences he has gained over the past five years to lead a successful career as an independent researcher. iii ACKNOWLEDGEMENTS First, I would like to express gratitude to my advisor, Paul F. Agris, for helping me to develop into a researcher. His mentoring and discussions about the inner workings of a lab and research facility have opened my eyes to the nuances that must be considered when starting a lab and the joys associated with the process. Also, my advisory committee has offered meaningful insights and knew how to offer constructive criticism to ensure that my projects were comprehensive. In particular, I acknowledge the willingness of E. Stuart Maxwell for opening his lab to me during my first year and offering his graduate student, Keith Gagnon, to teach me cloning techniques. I extend gratitude to the faculty, staff and graduate students of the Department of Molecular and Structural Biochemistry at North Carolina State University and the Life Sciences Building at the University at Albany for their friendship, advice and willingness to offer their expertise. No acknowledgement would be complete without noting the contributions of current Agris lab members and alumni. I would like to specifically thank William Graham, Franck Vendeix, Estella Gustilo, Yann Bilbille and Kun Lu for their hard work and contributions to my projects. I would also like to thank the undergraduates that I have had the opportunity to mentor and have assisted with various aspects of my projects: Jia Kim, Minhal Makshood and Erick Harr. My friendships and experiences over the past five years with members of the Agris lab in both Raleigh and Albany have shaped who I am as a researcher and I owe a great deal of my successes to them. Finally, I thank my loving wife. Not only has she supported my research and acted as a constant inspiration to me, but she has been unwavering in her willingness to help me overcome all obstacles during my graduate career. From staying by my side during a brief medical scare to leaving her job and moving with me to New York, she has gone above and beyond what I could have hoped for. iv TABLE OF CONTENTS LIST OF TABLES ............................................................................................................... viii LIST OF FIGURES ............................................................................................................... ix CHAPTER 1. Introduction and literature review ............................................................... 1 1.1 Transfer ribonucleic acid (tRNA): functions and modification ..................................... 1 1.2 Anticodon domain modifications ................................................................................... 3 1.2.1 Position 34 modifications ....................................................................................... 3 1.2.2 Position 37 Modifications ....................................................................................... 5 1.2.3 Position 32 Modifications ....................................................................................... 6 1.3 Modulation of anticodon stem and loop structure, stability and function ..................... 7 1.4 Six-fold degenerate decoding of arginine in Escherichia coli ....................................... 9 1.5 References .................................................................................................................... 12 1.6 Tables and Figures ....................................................................................................... 20 CHAPTER 2. Modifications modulate anticodon loop dynamics and codon recognition of E. coli tRNAArg1,2 ............................................................................................................... 27 2.1 Statement of project contribution................................................................................. 27 2.2 Abstract .......................................................................................................................