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Requirements and Rationale for Amber Translation As Pyrrolysine Dissertation

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Requirements and Rationale for Amber Translation As Pyrrolysine Dissertation REQUIREMENTS AND RATIONALE FOR AMBER TRANSLATION AS PYRROLYSINE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By David Gordon Longstaff, B.Sc. (Hons), M.S. ***** The Ohio State University 2007 Dissertation Committee: Dr. Joseph A. Krzycki, Adviser Approved by Dr. Charles J. Daniels Dr. John N. Reeve Dr. F. Robert Tabita Adviser Graduate Program in Microbiology ABSTRACT Methanosarcina spp. are capable of utilizing methylamines as growth substrates for methanogenesis. The methylamine methyltransferases responsible for initiating this process contain an unusual amino acid, pyrrolysine, encoded by an in-frame amber codon. This dissertation examines how pyrrolysine has infiltrated the genetic code of Methanosarcina acetivorans, and the rationale for this amino acid being maintained. Amber translation as pyrrolysine requires a specific pyrrolysyl-tRNA synthetase, encoded by pylS, that catalyzes the ligation of pyrrolysine onto an amber-decoding tRNA (tRNApyl), encoded by pylT. Expression of the pylT and pylS genes in E. coli, in the presence of an exogenous source of pyrrolysine, is sufficient to allow amber translation as pyrrolysine in this organism (Chapter 2). In the Methanosarcina spp., three other genes, pylB, pylC, pylD, have been identified that are thought to be co-transcribed with pylTS. Co-expression of pylBCD with pylT and pylS in E. coli allowed amber translation as pyrrolysine to occur in the absence of an exogenous source of pyrrolysine, showing that PylB, PylC and PylD play a role in pyrrolysine biosynthesis. This suggests the pyl operon acts as a genetic code expansion cassette allowing the transmissible genetic encoding of pyrrolysine (Chapter 3). ii A major question for amber translation as pyrrolysine was whether a cis-acting element, dubbed the pyrrolysine insertion sequence PYLIS, was required. Recombinant expression of the monomethylamine methyltransferase gene, mtmB, in the native organism Methanosarcina acetivorans allowed this problem to be addressed. In the absence of PYLIS, amber-translation as pyrrolysine was still observed generating approximately 30% of the full-length product compared to wild-type levels. Whilst showing PYLIS is not essential of UAG translation, the decrease in production of full- length methylamine methyltransferases would be problematic for the methanogen, which produces little energy from methanogenesis (Chapter 4). This system has enabled functional studies to be performed on MtmB by allowing the replacement of pyrrolysine, and residues within hydrogen-bonding distance of pyrrolysine, with other amino acids by site-directed mutagenesis. These replacements lead to a loss of MtmB activity, whilst maintaining the ability to bind the cognate corrinoid protein, MtmC. Combined, this suggests that pyrrolysine is essential for methanogenesis from methylamines (Chapter 5). iii Dedicated to my wife Heather, and to my parents Linda and David, whose support and encouragement has been invaluable throughout my studies iv ACKNOWLEDGMENTS I wish to thank my advisor, Joseph Krzycki, and my committee members John Reeve, Charles Daniels and F.Robert Tabita for their wisdom, support and patience that aided me greatly in completing this thesis. I would like to further extend my thanks to the other members of the Krzycki lab, whose friendship and support have been invaluable, and who have also provided materials and insight into these projects. In particular I would like to thank the following: Jitesh Soares for his generosity in providing me with the MtbA and RAM proteins, and his patience in teaching me the coenzyme M methylation assay. Sherry Blight and I worked together to elucidate the role of the PYLIS element in UAG translation as pyrrolysine, without her contributions this work would have taken years longer. Ross Larue has provided low molecular weight cell extracts from Methanosarcina Spp. and E. coli for detection by the colorimetric reporter system discussed in chapter 2. Ross Larue and Anirban Mahapatra also worked to provide in vitro evidence confirming pyrrolysine biosynthesis in E. coli harboring the pylBCD genes [Chapter 3]. I would also like to thank the following for help with anaerobic mass-culturing and harvesting of the methanogens: Brian Martin, Steve Smith, Josephine Wang, Taminder Singh and Todd Matulnik for harvesting the cells anaerobically. v VITA November 13, 1978 …………………..Born – Leicester, England 2001 …………………………………. B.Sc. (hons) Microbiology and Genetics, University of Aberdeen, Scotland 2007 …………………………………. M.S. Microbiology, The Ohio State University 2001 – 2007 …………………………. Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS Research Publication 1. Blight, S.K., Larue, R.C., Mahapatra, A., Longstaff, D.G., Chang, E., Zhao, G., Kang, P.T., Green-Church, K.B., Chan, M.K., and Krzycki, J.A. (2004) Direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo. Nature 431: 333- 335. 2. Longstaff, D.G., Blight, S.K., Zhang, L., Green-Church, K.B., and Krzycki, J.A. (2007) In vivo contextual requirements for UAG translation as pyrrolysine. Mol Microbiol 63: 229-241. vi 3. Longstaff, D.G., Larue, R.C., Faust, J.E., Mahapatra, A., Zhang, L., Green- Church, K.B., and Krzycki, J.A. (2007) A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine. Proc Natl Acad Sci U S A 104: 1021-1026. FIELDS OF STUDY Major Field: Microbiology vii TABLE OF CONTENTS Page Abstract…………………….…………………………………………….…….. ii Dedication………………………………………………………………….…... iv Acknowledgments……………………………………………………………… v Vita………………………………………………………………………….….. vi List of Tables……………………………………….…………………………... xii List of Figures…………………………………………………………………... xiv List of Symbols and Abbreviations…………………………………………….. xviii Chapters: 1. Introduction…………………………………………………………. 1 1.1 The genetic code………………….……………………... 1 1.2 Identification of the 22nd amino acid, pyrrolysine............. 3 1.3 Translation………………………………………………. 12 1.4 Aminoacyl-tRNA synthetases…………………………… 13 1.5 Translation initiation…………………………………….. 16 1.6 Translation elongation…………………………………… 21 1.7 Translation termination………………………………….. 22 1.8 Deviations from the standard genetic code………...……. 25 1.9 Theories of codon reassignment…………………………. 26 1.10 Frameshifting…………………………………………….. 28 1.11 Ribosomal hopping………………………………………. 31 1.12 The 21st genetically encoded amino acid, selenocysteine.. 32 1.13 Overview of thesis……………………………………….. 35 2. Incorporation of pyrrolysine into proteins expressed in Escherichia coli 2.1 Introduction……………………………………………… 37 2.2 Materials and Methods………………………………….. 42 2.2.1 Construction of plasmids………………………… 42 2.2.2 Sources of L-pyrrolysine – chemically synthesized or enriched low molecular weight fraction from Methanosarcina barkeri…… .....……………....... 44 viii 2.2.3 Assay conditions to monitor pyrrolysine dependent amber suppression in Escherichia coli…..……… 45 2.2.4 Isolation of recombinant MtmB1 synthesized by E. coli for mass spectrometric analysis…………. 46 2.3 Results…………………………………………………… 47 2.3.1 The UAG in mtmB1 is translated in E. coli bearing pyl/S and pylT in the presence of pyrrolysine…... 47 2.3.2 Determination of the residue mass at the position encoded by UAG in MtmB1 synthesized by E. coli 52 2.3.3 UAG translation in mtmB1 is correlated to the concentration of supplied pyrrolysine between the ranges of 20 – 1000 µM…………………………. 57 2.3.4 Incorporation of pyrrolysine into β-glucuronidase 60 2.4 Discussion……………………………………………….. 74 3 Expansion of the genetic code of Escherichia coli to include endogenously biosynthesized pyrrolysine 3.1 Introduction………………………...…………………… 79 3.2 Materials and Methods…………………………………. 83 3.2.1 Construction of pyl gene expression constructs…. 83 3.2.2 Construction of reporter plasmids……………….. 84 3.2.3 Expression of recombinant mtmB1 in Escherichia coli containing pyl genes………………………… 88 3.2.4 Analysis of amber suppression of an in-frame UAG codon in uidA expressed in Escherichia coli……. 88 3.2.5 Determination of the mass of the residue at the UAG-encoded position in recombinant MtmB1.... 89 3.3 Results…………………………………………………… 90 3.3.1 UAG translation in Escherichia coli is enhanced upon expression of pylT and pylS with the pylBCD genes…………………………………………….. 90 3.3.2 All five pylTSBCD genes are required for UAG translation in E. coli…………………………….. 104 3.3.3 Pyrrolysine is biosynthesized by PylB, PylC and PylD in E. coli…………………………………… 106 3.3.4 Translation of a UAG codon introduced into the E. coli uidA gene by pylTSBCD…………………. 110 3.4 Discussion……………………………………………….. 113 ix 4 In vivo contextual requirements for UAG translation as pyrrolysine in Methanosarcina acetivorans C2A………………………………….. 120 4.1 Introduction……………………………………………... 120 4.2 Materials and Methods………………………………….. 124 4.2.1 Construction of the C–terminal hexahistidine tagged MtmB1 vector, pDL05C………………………… 124 4.2.2 Constructions of the N–terminal hexahistidine tagged MtmB1 vector, pDL05N………………………… 125 4.2.3 Mutation of the amber codon in pDL05C and pDL05N to either TAA or GCA………………… 127 4.2.4 Replacement of the PYLIS element with sequence from the structurally homologous MetH………… 128 4.2.5 Liposome mediated transformation into Methanosarcina acetivorans…………………….. 129 4.2.6 Immunoblotting………………………………….. 130 4.3 Results……………………………………………………. 131 4.3.1 Expression of recombinant mtmB1 in Methanosarcina acetivorans does not require the 5’ or 3’ untranslated regions…………………………………………….
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