Old Dominion University ODU Digital Commons Theses and Dissertations in Biomedical Sciences College of Sciences Summer 1997 The Cellular and Molecular Dynamics of the Queuosine Modification in rT ansfer RNA: Definition, Modulation, Deficiencies and Effect of the Queuosine Modification System Rana C. Morris Old Dominion University Follow this and additional works at: https://digitalcommons.odu.edu/biomedicalsciences_etds Part of the Biochemistry Commons, Biophysics Commons, Molecular Biology Commons, and the Oncology Commons Recommended Citation Morris, Rana C.. "The Cellular and Molecular Dynamics of the Queuosine Modification in rT ansfer RNA: Definition, Modulation, Deficiencies andff E ect of the Queuosine Modification System" (1997). Doctor of Philosophy (PhD), Dissertation, , Old Dominion University, DOI: 10.25777/bwhj-g976 https://digitalcommons.odu.edu/biomedicalsciences_etds/63 This Dissertation is brought to you for free and open access by the College of Sciences at ODU Digital Commons. It has been accepted for inclusion in Theses and Dissertations in Biomedical Sciences by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. THE CELLULAR AND MOLECULAR DYNAMICS OF THE QUEUOSINE MODIFICATION IN TRANSFER RNA: DEFINITION, MODULATION, DEFICIENCIES AND EFFECT OF THE QUEUOSINE MODIFICATION SYSTEM by Rana C. Morris B.S. May 1990, the College of William and Mary in Virginia A Dissertation Submitted to the Faculties of Old Dominion University and Eastern Virginia Medical School in Partial Fulfillment of the Requirement for the Degree of DOCTOR OF PHILOSOPHY BIOMEDICAL SCIENCES OLD DOMINION UNIVERSITY and EASTERN VIRGINIA MEDICAL SCHOOL August 1997 Approved by: irk S. Elliott (Chair) rank J. Castora hristophensL^Osgeod Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT THE CELLULAR AND MOLECULAR DYNAMICS OF THE QUEUOSINE MODIFICATION IN TRANSFER RNA: DEFINITION, MODULATION, DEFICIENCIES, AND EFFECT OF THE QUEUOSINE MODIFICATION SYSTEM Rana C. Morris Old DominioQ University and Eastern Virginia Medical School, 1997 Director Dr. Mark S. Elliott The presence of the queuosine modification in the wobble position of tRNA“ , tRNA*5*, tRNAhIS, and tRNA151* is associated with a decrease in cellular growth rate, an increase in the ability to withstand environmental stress, and differentiation of pleuripotent cells into mature phenotypes. The loss of this normal modification is strongly correlated with neoplastic transformation and tumor progression of a wide variety of cancers. The “normal” system for formation of the queuosine modification in tRNA was studied in human fibroblast cell cultures and in mouse, rat and human liver tissues. The queuosine modification system is shown to be made up of three distinct mechanisms: uptake of the queuine base across the plasma membrane; incorporation of this base into cytoplasmic tRNA; and salvage of the base from products of normal tRNA degradation. The queuine membrane transporter and incorporation enzyme are activated via phosphorylation by protein kinase C and inactivated by the action of a phosphatase. This regulation by phosphorylation integrates the queuosine modification system into a very sensitive eukaryotic cellular switching mechanism already known to produce phenotypic alterations with strong correlations to changes in queuosine levels. A comparative study of two “abnormal” human adenocarcinoma cell-lines (colon and breast) was performed to assess their queuosine levels and determine the malfunctioning system step(s) for the cause of the observed deficiencies. The 100% queuosine-deficient colon tumor cell-line possessed a null mutation for the queuosine incorporation enzyme, while the 50-60% queuosine-deficient breast tumor cell-line exhibited a strong deficiency in the queuine salvage mechanism. These results demonstrate the potential for determination of even multiple sites of lesions in the modification sytem that would yield queuosine- deficient tRNA characteristic of tumors. Computational modeling was utilized to determine the biological function for the queuosine modification. Steric, electrostatic, and structural differences were observed for queuosine, queuosine- analogues and guanosine, the nucleosides incorporated into tRNA“p anticodon stem/loop structures, and in triad complexes of tRNA*sp with mRNA and tRNA1*®. The results of this research identify indistinguishable energetic parameters for complexes of queuosine-modified anticodon loops when paired with an mRNA containing cytosine- or a uridine-ending codon. However, guanosine-containing anticodon loops demonstrate much stronger energetic stability with cytosine-ending codons. The difference in codon Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bias is shown to be due to the restriction of anticodon loop flexibility by a queuosine-induced extended intraloop hydrogen bonding network and only minimally due to a shift in hydrogen bonding pattern produced by an intraresidue hydrogen bond. A key difference in the physiology of normal and neoplastic cells is in the increased expression of oncodevelopmental genes with respect to those housekeeping genes needed for survival. Sequence analysis of several oncodevelopmental and housekeeping transcripts suggests the presence of a contrasting bias in the usage of queuosine*related codons which end in cytosine or either cytosine or uridine, respectively. In combination with the mechanism proposed for tRNA 19 decoding preferences, this codon usage bias suggests a potentially influential role for the queuosine modification system in the translational control of oncodevelopmental gene expression. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ‘Turns out not where but who you’re with that really matters. ‘Turns out not where but what you think that really matters. Whatever tears at us, whatever holds us down, if nothing can be done we’U make the best of what’s around. paraphrased from “The Best of What’s Around” by the Dave Matthews Band from their Album Under the Table and Dreaming (1994, The RCA Records Label) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS As much as I've toiled over the preparation of this document, I have procrastinated and postponed writing this “Acknowledgments” section for last because there is no way to adequately compensate the large number of extremely supportive people who have come into my life during my tenure in graduate school. Through the years of agonizing over coursework, written and oral comprehensive exams, research proposals, research itself an oral defense of the research, and the preparation of this enormous document, there have been many people who have contributed to my success, as well as intellectual and emotional survival. I extend my heartfelt gratitude to a large number of people, only some of whom are listed below. Foremost in their tolerance and patience have been my family. To the person who most strongly supported me throughout my tenure in graduate school and in life, I thank Jim for absolutely everything. There is not much else I can say to express my appreciation for your encouragment To the two little rugrats (Jamie and Sean! who showed me that there are truly important things in life, and that distraction, oftimes, can provide perspective that can solve even the most insurmountable of problems, I offer my biggest hugs and kisses. My mother and father (Steve and Louise), sister (Jo) and both sets of grandparents (Naomi & Sim and Ruth & Mannv! have well practiced their heritage of affectionate nagging for tutorials, seminars, reprints and official graduation dates. Even my father-in-law (Jim. Jr.) learned the art of the nag, though not through genetic inheritance. But their love and devotion saw me through the endless reschedulings and postponements. To the only other person who has had to deal with me day in and day out, in coffee and M-n-M surplus and in drought, through boring, incomprehensible seminar and defense, and with new creative ideas that work and that fail, I recognize the tireless, wit-filled, optimistic support from Dr. Mark S. Elliott my research advisor and cohort. However, an additional bucket-o-gratitude must also be extended to his family (Becky. Brent Dave, and Matt..not to mention the three does, rabbit and hamsterl who tolerated his absences and frustrations due to lab problems and successes. My many thanks to my Dissertation Committee (Dr. Frank Castora. Dr. Chris Osgood, and Dr. James Yuan) for their patience, suggestions and guidance, and above all for the time spent reviewing this manuscript. Many faculty members within the Department of Biochemistry and Chemistry aided me not only in my academic education, but also in my professional and personal development. Among these extremely supportive folks are: Dr. Charles Bell. Dr. Ken Brown. Dr. Bob Ake. Dr. Allen Clark. Dr. Pat Pleban. Dr. Laura Moen. Dr. Rov Williams, and Dr. Keith Carson from the Biology Department The large number of students who have come and gone over the years have been encouraging in their willingness to help, provide useful suggestions and criticisms, or just lend a patient ear. I thank a few in particular (soon-to-be! Dr.s Thomas Heard. Miguel Santos and Lester Pretlow. (already) Dr.s Jackie Smith and Sandi Ward, and Julie Patrick.