University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2003-05-08 Peptidyltransfer Reaction Catalyzed by the Ribosome and the Ribozyme: a Dissertation Lele Sun University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Cells Commons, Hormones, Hormone Substitutes, and Hormone Antagonists Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, Pharmaceutical Preparations Commons, and the Therapeutics Commons Repository Citation Sun L. (2003). Peptidyltransfer Reaction Catalyzed by the Ribosome and the Ribozyme: a Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/6ds0-4c54. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/115 This material is brought to you by eScholarship@UMMS. 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PEPTIDYL TRANSFER REACTION CATALYZED BY THE RIBOSOME AND THE RIBOZYME A Dissertation Presented Lele Sun Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY BIOCHEMISTRY AND MOLECULAR BIOLOGY (May 8, 2003) PEPTIDYL TRANSFER REACTION CATALYZED BY THE RIBOSOME AND THE RIBOZYME A Dissertation Presented By Lele Sun Approved as to style and content by: Dr. Tony Ip, Chair of Committee Dr. Sharon Huo, Member of Committee Dr. David Lambright, Member of Committee Dr. Craig Mello, Member of Committee Dr. Thoru Pederson, Member of Committee Dr. Biliang Zhang, Dissertation Mentor Dr. Anthony Carruthers, Dean of the Graduate School of Biomedical Sciences Interdisciplinary Graduate Program May 8, 2003 ii COPYRIGHTS Chapter II is copyrighted by: Lei Zhang , Lele Sun , Zhiyong Cui, Robert L. Gottlieb, Biliang Zhang. (2001) 5' Sulfhydryl-modified RNA: initiator synthesis, in vitro transcription, and enzymatic incorporation. Bioconjugate Chemistry , 939-948. Chapter III is partially copyrighted by: Lele Sun, Zhiyong Cui, Robert L. Gottlieb, Biliang Zhang. (2002) A selected ribozyme catalyzing diverse dipeptide synthesis. Chemistry Biology 619-628. Chapter V is partially copyrighted by: Biliang Zhang, Lei Zhang, Lele Sun, Zhiyong Cui. (2002) Synthesis ofpCpCpA- NH- phenylalanine as a ribosomal substrate. Organic Letters 4 (21), 3615-3618. ) These authors contribute equally to the work. ACKNOWLEDGEMENTS First of all, I would like to thank my mentor, Dr. Biliang Zhang for his comprehensive support and scientific instruction throughout the thesis research. I thank Dr. Zhiyong Cui for the excellent chemical synthesis of the compounds in all the experiments I have conducted. I thank Dr. Lei Zhang for his contribution to the synthesis ofCCA-NH-Phe substrate. I thank Chunfang Li for helping me perform part of the kinetics experiments. I thank the research advisory and defense committee for their helpful comments on my research. I thank Dr. Anna Delprato and Dr. Heather Cook for reading the whole manuscript and for their helpful comments. Finally, I appreciate all the people that have helped me during the thesis research. ABSTRACT The "RNA world" hypothesis makes two predictions that RNA should have been able both to catalyze RNA replication and to direct protein synthesis. The evolution RNA-catalyzed protein synthesis should be critical in the transition from the RNA world to the modem biological systems. Peptide bond formation is a fundamental step in modem protein biosynthesis. Although many evidence suggests that the ribosome is a ribozyme, peptide bond formation has not been achieved with ribosomal RNAs only. The goal of this thesis is to investigate whether RNA could catalyze peptide bond formation and how RNA catalyzes peptide bond formation. Two systems have been employed to approach these questions, the ribozyme system and the ribosome system. Ribozymes have been isolated by in vitro selection that can catalyze peptide bond formation using the aminoacyl-adenylate as the substrate. The isolation of such peptide- synthesizing ribozymes suggests that RNA of antiquity might have directed protein synthesis and bolsters the "RNA world" hypothesis. In the other approach, a novel assay has been established to probe the ribosomal peptidyltransferase reaction in the presence of intact ribosome, ribosomal subunit, or ribosomal RNA alone. Several aspects of the peptidyltransfer reaction have been examined in both systems including metal ion requirement, pH dependence and substrate specificity. The coherence between the two systems is discussed and their potential applications are explored. Although the ribozyme system might not be a reminiscence of the ribosome catalysis, it is still unique in other studies. The newly established assay for ribosomal peptidyltransferase reaction provides a good system to investigate the mechanism of ribosomal reaction and may have potential application in drug screening to search for the specific peptidyltransferase inhibitors. .:. .................... .. .. .. .. .. .. .. .. .. .. .. ... .................................................................... ... ... ... ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .............................. .. .. .. .. .. .. .. .. .. .. ... ... .. ........... .. ... ... ... ... .. .. .. .. .. .... .. ........ TABLE OF CONTENTS Title Page. Approval Signatures.... .ii Copyrights......................................................................... ...... iii Acknowledgements. Abstract. ... v Table of Contents................................................................................ .... List of Figures .ix List of Tables. Chapter I: Introduction.............................................................................. Chapter II: 5' Sulfhydryl-Modification ofRNA --- An Application in Studying RNA Structure and Function.............................................................................. Experimental Procedures.... Conjugation of 5' HS-PEG GMP-RNA with maleimide-activated HRP ......... Thiol-reactive biotin conjugation with 5' HS-PEG GMP-RNA ................... Thiol-reactive biotin conjugation with 5' HS- RNA Conclusion...... Chapter III: Peptide-Synthesizing Ribozymes Using the Aminoacyl-Adenylate as the Substrate............................................................................................ Experimental Procedures. In vitro selection ofthe R180 ribozyme family..................................... ... .:..:..:..:..:..:. .. ..... .......... ... .... ....................... ............................................... ........... .... .. .. ... .. ............................................. .......... ... .. ."""""" .. .................. ....... .. .. ........... .. .. .... .. ...... .. .. ......... .. ................... ............... .... ........... .. ........... .. ............. .. .... ..... .... .. .... .... Secondary structure model of the Rl80 family.... .. .. Reaction and product validation ofR180 catalysis The aminoacyl-adenylate as the substrate for a previous isolated peptide- synthesizing ribozyme (the C25 ribozyme) Amino acid specificity for the R 180 and C25 ribozymes ............................. Distinguished recognition of AMP by the R180 and C25 ribozymes Long peptide synthesis by the Rl80 and C25 ribozymes Conclusion................................................................................ Chapter IV: Characterization of the R180 Catalysis: Metal Ion Requirement, pH Dependence and Kinetics....................................................................... ... Experimental Procedures. .40 Trimolecular reaction of dipeptide synthesis.. .44 Metal requirement. .46 pH dependence & isotope effects............................... ...................... .. Kinetics analysis........................................................................ ... Conclusion................................................................................ Chapter V: A Novel Assay For Ribosomal Peptidyltransferase Activity.............. .... Experimental Procedures.............................................................. ... A novel assay for ribosomal peptidyltransferase activity........................... Divalent metal ion specificity.............. pH dependence....... ... ... ...... ... P-site substrate specificity. ...... Antibiotics inhibition studies........................................................ ..... V11 .:. ..... ..... Conclusion................................................................................ Chapter VI: Summary and Discussion.. References......................................................................................... .. V11 ...................................................................................................... .. .............. .. """"""""" .. .. .. .. ............. ......... .... .. LIST OF FIGURES Figure 1- 1. Reactions catalyzed by ribozymes .................................................. Figure 1-2. General scheme of in vitro selection and evolution.............................. Figure 1- 3. Crystal structures of the large ribosomal subunit.. .. .. .. 5 Figure 2- 1. Chemical structures ofHS-PEGz-GMP, HS-PEG GMP, and GSMP ..... .. Figure 2-2. Autoradiogram ofRNAs transcribed in the presence and absence