I: Expanding Genetic Code in S. cevevisiae with an Orthogonal tRNATrp/UCA/ Tryptophanyl tRNA Synthetase Pair II: Synthetic Yeast Prions based on a Non-NQ-rich Amyloidogenic Sequence Derived from the NAC Protein Sequence of α-Synuclein. Open Access

Abstract

In an approach to expand the genetic code, a general strategy has been developed to utilize an orthogonal tRNA and aminoacyl-tRNA-synthetase (aaRS) pair. Given the high homology of the translational machinery for eukaryotic cells, Saccharomyces cerevisiae is an ideal gateway host to develop an orthogonal pair for expansion of the genetic code in eukaryotic organisms. This process will facilitate the incorporation of biophysical probes into proteins for biophysical analysis or provide another protein evolution pathway. Therefore, in Chapter 2 of this thesis, we describe the methodology of constructing an orthogonal Mycoplasma genitalium tryptophanyl-tRNA synthetase (MgenTrpRS)/MgentRNA^Trp pair for incorporation of non-canonical amino acids in S. cerevisiae cell in response to opal (UGA) codon. In chapter 3, a library selection scheme to identify mutant MgenTrpRS variants with altered selectivity for the non-canonical 5-hydroxy-tryptophan and 5-methoxy-tryptophan is investigated and the spectroscopic properties of enhanced cyan fluorescent protein (ECFP) containing these non-canonical amino acids in the chromophore are described.

Studies of the yeast prions [PSI⁺] and [URE3] have revealed the mechanism of prion formation and propagation in S. cerevisiae. It was recently suggested that that substituting the N/Q-rich subdomain with other high Q/N content sequences can construct novel yeast prions. The interchangeability of the NQ domain with other aggregation-prone sequences not only helps illuminate the principles of yeast prion domain architecture, but also allows prion [PSI⁺] to serve as a model to explore the controllable epigenetic regulators. In Chapter 4, a non-Aβ-component (NAC) fragment from α-synuclein, which lacks the compositional bias towards Gln/Asn that is characteristic of the yeast prion domains described thus far, is explored as a structural alternative for the NQ domain of Sup35p to generate a novel chimeric yeast prion. The results demonstrated that a novel prion [NAC⁺] could arise from domain substitution within Sup35p, and that the sequence of the prion-forming domain was not restricted to Asn/Gln-rich proteins. This model system might be useful for investigation of the potential for in vivo transmission of amyloidogenic sequences associated with protein mis-folding diseases.

Table of Contents

Table of Contents
CHAPTER Page
Chapter I: Introduction.................................................................................................... 1

Expanding the Genetic Code: Adding Unnatural Amino Acids into Proteins ................ 2
Design of a chimeric yeast prion in S. cerevisiae ......................................................... 13
Reference ...................................................................................................................... 22
Chapter II: Creation of an orthogonal tRNA^Trp/UCA/ tryptophanyl tRNA synthetase
pair for incorporation of amino acid analogues in S. cevevisiae ................................. 32
Introduction ................................................................................................................... 33
Experimental Procedures .............................................................................................. 40
Results and Discussion ................................................................................................. 49
An orthogonal MgentRNA^Trp/UCA for S. cerevisiae................................................... 49
In vivo aminoacylation assay confirms the MgentRNA^Trp/UCA / MgenTrpRS pair is
orthogonal in S. cerevisiae ........................................................................................ 50
The MgenTrpRS specifically recognize the MgentRNA^Trp/UCA for the incorporation
of Tryptophan to the UGA mutation on the Trp66 of ECFP .................................... 51
Optimizing the suppression efficiency of the MgentRNA^Trp /MgenTrpRS pair ....... 53
Conclusion .................................................................................................................... 58
Reference ...................................................................................................................... 76
Chapter III: Development of a Screening System for the Expanding Genetic Code in
Yeast to Incorporate Tryptophan Analogues ............................................................... 81

Introduction ................................................................................................................... 82
Experimental Procedures .............................................................................................. 85
Result and Discussion ................................................................................................... 99
Analysis of the screening effect of ura3-14 reporter gene ....................................... 99
Site-saturation mutagenesis of MgenTrpRS ........................................................... 101
Library screening of MgenTrpRS mutants with activity toward 5-OH-Trp ........... 103
Library screening of MgenTrpRS mutants with activity toward 5-MeO-Trp ......... 106
Spectral analysis of ECFP and UAA incorporated ECFP variants ......................... 107
Confocal microscopy image analysis of cells expressing ECFP and UAA
incorporated ECFP variants .................................................................................... 108
Conclusion .................................................................................................................. 108
Reference .................................................................................................................... 127
Chapter IV: Synthetic Yeast Prions Based on a Non-NQ-rich Amyloidogenic
Sequence Derived from the NAC Protein Sequence of α-Synuclein. ....................... 131
Introduction ................................................................................................................. 132
Experimental Procedures ............................................................................................ 135
Results ......................................................................................................................... 150
Cells containing the SYN1-G-Sup35 and NAC-G-Sup35 fusion proteins both
showed two phenotypes .......................................................................................... 150
Aggregation state of NACp and SYN1p are different ............................................ 152

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Keyword	Genetic Code Prion Yeast protein misfolding tryptophan
Committee Chair / Thesis Advisor	Conticello, Vincent, Emory University
Committee Members	Lynn, David, Emory University Edmondson, Dale E, Emory University

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