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Huang Gsas.Harvard 0084L 11365 Study on Bacterial Protein Synthesis System toward the Incorporation of D-Amino Acid & Synthesis of 2'-deoxy-3'-mercapto-tRNA The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Huang, Po-Yi. 2014. Study on Bacterial Protein Synthesis System toward the Incorporation of D-Amino Acid & Synthesis of 2'- deoxy-3'-mercapto-tRNA. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12274132 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Study on Bacterial Protein Synthesis System toward the Incorporation of D-Amino Acid & Synthesis of 2’-deoxy-3’-mercapto-tRNA A dissertation presented by Po-Yi Huang to Department of Chemistry & Chemical Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Chemistry Harvard University Cambridge, Massachusetts April, 2014 ) © 2014 by Po-Yi Huang All rights reserved. Dissertation advisor: Dr. George Church Po-Yi Huang Study on Bacterial Protein Synthesis System toward the Incorporation of D- Amino Acid & Synthesis of 2’-deoxy-3’-mercapto-tRNA Abstract Life is anti-entropic and highly organized phenomenon with two characteristics reinforcing each other: homochirality and the stereospecific catalysis of chemical reactions. The exclusive presence of L-amino acids and R-sugars in living world well depict this. Hypothetically, the amino acids and sugars of reverse chirality could form a parallel kingdom which is highly orthogonal to the present world. The components from this mirror kingdom, such Ò as protein or nucleic acid, will be much more resistant to the defensive mechanism of present living system, which could be of great value. Therefore, by gradually rewiring the present bio- machineries, we look to build a bridge leading us to the space of mirror-imaged biomolecules. We begin by investigating protein synthesis with mirror amino acid since most amino acids contain one chiral center to be inversed comparing to sugars. In this work, we analyzed three stages critical for the incorporation of D-amino acid into ribosomal protein synthesis: amino acylation, EF-Tu binding of amino acyl-tRNA and delivery bias, and ribosome catalyzed peptidyl transfer. We have demonstrated that the affinity between EF-Tu and amino acyl-tRNA plays critical role on D-amino acid incorporation, and built a platform aimed to select for ribosome tolerating D-amino acid better. iii Non-ribosomal peptide and polyketide synthesis are another important class of modular biomolecules synthesis. Polyketides, which compose one of the largest groups of therapeutic natural products known today, are built by series of modular polyketide synthetases in bacteria, fungi and plants. Intrigued by their interaction with critical pathways in the cell and hence their therapeutic value, researchers have synthesized numerous polyketides either one by one or in a combinatorial manner. However, both these approaches become inefficient when a large set of scaffold-variant libraries of polyketide is required for drug development. In an effort to address this issue, we propose to utilize a bacterial translation system to build polyketide or polyketide- peptide hybrid scaffolds. More specifically, tRNA with a thiol in place of the hydroxyl group at the 3’-terminus is synthesized, loaded with malonyl ketide substrate and subjected into an in vitro translation system. ) iv Table of Contents CHAPTER 1: INTRODUCTION ............................................................................................... 1 THE SELF-SUSTENTION OF BIOLOGICAL CHIRALITY AMONG AMINO ACID AND SUGAR ENANTIOMERS . 2 HOW DO CELLS INTERACT WITH D-AMINO ACIDS AND L-SUGARS? ............................................................. 4 RIBOSOMAL INCORPORATION OF D-AMINO ACIDS INTO PEPTIDES .............................................................. 6 MUTAGENESIS AND SELECTION OF RIBOSOME FOR THE ABILITY TO INCORPORATE D-AMINO ACID CONSECUTIVELY .......................................................................................................................................... 9 NON-RIBOSOMAL PEPTIDE AND POLYKETIDE SYNTHESIS .......................................................................... 11 REFERENCES .............................................................................................................................................. 12 CHAPTER 2: FROM D-AMINO ACID TO D-AMINOACYL-TRNA AND ITS INCORPORATION TO PEPTIDE THROUGH EF-TU BINDING .................................... 17 SUMMARY .................................................................................................................................................. 18 INTRODUCTION .................................................. ......................................................................................... 18 RESULTS .................................................. ................................................................................................... 19 Specificity of aminoacyl-tRNA synthetases .............................................................................................................. 19 Non-enzymatic acylation and chemical acylation ..................................................................................................... 23 Two assays for D-amino acid incorporation and the interaction) of aa-tRNA and EF-Tu .......................................... 27 Mutagenesis study of EF-Tu for enhanced D-AA delivery capacity ......................................................................... 34 DISCUSSION .................................................. .............................................................................................. 38 MATERIALS AND METHODS ....................................................................................................................... 40 REFERENCES .............................................................................................................................................. 45 CHAPTER 3: RIBOSOMAL ENGINEERING - MUTAGENESIS AND SELECTION ... 48 SUMMARY .................................................................................................................................................. 49 INTRODUCTION .................................................. ......................................................................................... 49 RESULTS .................................................. ................................................................................................... 50 Design and construction of library............................................................................................................................. 50 Selections with stepwise increasing stringency ......................................................................................................... 52 Sequence analysis and functional assay ..................................................................................................................... 54 DISCUSSION .................................................. .............................................................................................. 56 MATERIALS AND METHODS ....................................................................................................................... 58 Construction of rRNA plasmid library ...................................................................................................................... 58 Purification of ribosome library or individual mutant ............................................................................................... 60 Selection of mutants by ribosome display ................................................................................................................. 61 Sequence analysis ...................................................................................................................................................... 61 Western blot and translation activity assay ................................................................................................................ 62 REFERENCES .............................................................................................................................................. 62 CHAPTER 4: SYNTHESIS OF 2’-DEOXY-3’-MERCAPTO-TRNA SUBSTRATE FOR RIBOSOMAL POLYKETIDE SYNTHESIS ......................................................................... 64 v SUMMARY .................................................................................................................................................. 65 INTRODUCTION .................................................. ......................................................................................... 65 RESULTS .................................................. ................................................................................................... 67 Design and Synthesis of 2’-deoxy-3’-mercapto-tRNA .............................................................................................. 67 DISCUSSION .................................................. .............................................................................................. 70 MATERIALS
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