CLONING and SEQUENCING of the GENE for VALYL-Trna
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CLONING AND SEQUENCING OF THE GENE FOR VALYL-tRNA SYNTHETASE FROM BACILLUS STEAROTHERMOPHILUS by Nigel John Brand A dissertation submitted to the Imperial College of Science and Technology in candidature for the Diploma of Imperial College, and to the University of London in candidature for the degree of Doctor of Philosophy. Department of Chemistry Imperial college London SW7 2AZ September 1986 2 ABSTRACT This thesis describes the determination of the DNA sequence of the valS gene from the bacterium Bacillus stearothermophilus, which encodes the enzyme valyl-tRNA synthetase (ValRS). The gene was cloned from a genomic library of B. stearothermophilus DNA in the plasmid vector pAT153 by complementation of an E. coli mutant containing a temperature-sensitive lesion in the chromosomal copy of valS. The gene was shown to have been cloned from a comparison of the kinetic properties of the cloned gene product to those of native ValRS, purified from B. stearothermophilus. Fragments of the gene and its adjoining regions were ultimately subcloned into the bacteriophage vector Ml 3 and their DNA sequences were determined by the dideoxy chain-termination method. The individual DNA sequences were amalgamated into one contiguous sequence spanning 5 kilobases (kb). The DNA sequence was analysed and an open reading frame, sufficient to code for an 880-residue protein of Mr = 102,036, was identified. This was assigned to ValRS by a number of criteria. First, the pattern of codon usage for the frame was shown to correlate with codon preferences of other aminoacyl-tRNA synthetases. Second, the amino acid composition, predicted from a translation of the DNA sequence, agreed with that determined for a sample of purified, cloned ValRS. A spectrophotometric determination of the number of tryptophan residues in the enzyme also agreed with the predicted value. The primary sequence of the ValRS was compared with those of a number of other synthetases. Some significant homologies were found, notably the conservation of two sequences that have been implicated in binding ATP and the 3’ end of tRNA in other synthetases. A striking degree of homology (25-32%) with the isoleucyl-tRNA synthetase (IleRS) of Escherichia coli was found, representing the most extensive homology yet reported between two aminoacyl-tRNA synthetases from different bacterial species. This argues that the two enzymes may have evolved from a common progenitor. 3 ACKNOWLEDGEMENTS This work would not have been possible without contributions from a number of people. I should to thank my colleagues Thor Borgford and Tammy Gray for the hard work, enthusiasm and patience that it took to sequence "our gene". I am especially grateful to Robin Leatherbarrow and Wally Ward for their critical reading of the manuscript and for their many helpful comments. My thanks also to Mick Jones for introducing me to molecular biology in the first place, and to Jack Knill-Jones for synthesising all the sequencing primers and for his unstinting interest in the project. Lastly, thanks to Lesia Gojda for some excellent illustrations, to Glyn Millhouse for taking the photographs and to Gail Craigie for her help with the typing and layout of this tome. There are too many people (past and present) to mention, but special thanks to the following for their company, coffee and chat: Sue Cotterill, Tony Wilkinson, Jian-Ping Shi, Hugh Jones, Alison Campbell, Tim Wells, Katy Brown, Steve Delaney, Carlos Flores, Ishtiag Qadri, Paula Zard and Tom Purcell. Finally, my sincere thanks to my supervisor, Professor Alan Fersht, for his advice and continual interest at all stages in the project. The work presented in this thesis was funded by the Medical Research Council. 4 CONTENTS Abstract 2 Acknowledgements 3 Contents 4 List of Figures 11 List of Tables 14 CHAPTER 1. INTRODUCTION 1.1 Reasons for Studying the Aminoacyl-tRNA Synthetases 15 1.2 The Aminoacyl-tRNA Synthetases: a historical background 17 1.3 General Properties of the Aminoacyl-tRNA Synthetases 20 1.3.1 The Synthetases Catalyse a Fundamental Reaction 20 1.3.2 Structural Properties of the Synthetases 24 1.3.3 Implications for the Evolution of the Synthetases 25 1.3.4 Internal Repetitive Elements 28 1.3.5 Interactions between the Synthetases and tRNAs 30 1.4 The Fidelity of Aminoacylation by Aminoacyl-tRNA Synthetases 33 1.4.1 General Introduction 33 1.4.2 Simple Discrimination Against Dissimilar Amino Acids 34 1.4.3 Some Synthetases have Editing Mechanisms 36 1.5 Cloning and Expression of Aminoacyl-tRNA Synthetase Genes 40 1.5.1 The Impact of Genetic Engineering 40 1.5.2 Sequencing of Synthetase Genes 41 1.5.3 Diversity in the Structure of the Promoters of Synthetase Genes 41 1.5.4 The Expression of Synthetase Genes is Regulated in a Variety of Ways 44 1.5.5 Attenuation as a Means of Controlling 5 Expression of the pheS, T Operon 47 1.6 Structural and Functional Homologies Between Synthetases 50 1.6.1 Homologies at the Level of the Primary Sequence 50 1.6.2 Dissection of Synthetase Structure with Respect to Function 53 1.7 Towards a Study of the Functional Arrangement of the Valyl-tRNA Synthetase 56 CHAPTER 2. EXPERIMENTAL: GENERAL RECOMBINANT DNA METHODS 2.1 Media 58 2.2 Bacterial Strains 59 2.3 Enzymes 59 2.4 Cloning Vectors 59 2.5 Restriction Enzyme Digestions 61 2.5.1 Conditions for Digestion of Plasmid DNA 61 2.5.2 Separation of Restriction Fragments through Agarose Gels 61 2.5.3 Purification of DNA Fragments 64 2.6 Preparation of Phosphatased Vector DNA 65 2.7 B. stear other mophilus Gene Library 65 2.7.1 Construction of the Gene Library 65 2.7.2 Amplification of the Gene Library 66 2.8 Purification of Plasmid DNA 67 2.8.1 Large-scale Plasmid Preparation 67 2.8.2 Small-scale Plasmid Preparation 68 2.9 Ligations 69 2.10 Repairing Cohesive Ends with T4 DNA Polymerase 70 2.11 Digestion of Linearised DNA with Nuclease Bal3\ 70 2.12 Transformation of Competent E. coli 71 2.12.1 Preparation of Competent Cells 71 2.12.2 Transformation of E. coli DH5 71 2.12.3 Transformation of E. coli 236c 72 6 CHAPTER 3. EXPERIMENTAL: KINETIC ASSAYS AND PROTEIN PURIFICATION 3.1 Enzyme Assays 73 3.1.1 Materials 73 3.1.2 Active-Site Titration 74 3.1.3 Aminoacylation (Charging) Assay 75 3.2 SDS-Polyacrylamide Gel Electrophoresis 77 3.3 Purification of Valyl-tRNA Synthetase 78 3.3.1 A Modified Active-Site Titration for ValRS 78 3.3.2 Cell Culture and Preliminary Purification of ValRS 79 3.3.3 Precipitation with Ammonium Sulphate 80 3.3.4 DEAE-Sephacel Chromatography 81 3.3.5 FPLC-Chromatography 83 3.3.6 A Second Heat Step, Concentration and Storage of the Purified Enzyme 86 3.3.7 Discussion 86 3.4 Amino Acid Sequencing 89 3.5 Amino Acid Composition Determination 90 3.6 Determination of the Number of Tryptophan Residues in ValRS 90 CHAPTER 4. CLONING OF THE B. stearothermophilus valS GENE 4.1 Introduction 93 4.2 Cloning of the valS Gene from B. stear other mophilus by Complementation of a Temperature-Sensitive E. coli Strain 97 4.2.1 Amplification of the B. stearothermophilus Library in the Mutant Strain 97 4.2.2 Selection for valS Plasmids by Complementation 98 4.2.3 Characterisation and Sizing of pNB Plasmids 100 4.2.4 Discussion 103 4.3 Subcloning of valS from pNBl 103 4.3.1 Introduction 103 7 4.3.2 Subcloning Strategy 104 4.3.3 Selection of valS Subclones Complementation 107 4.3.4 Discussion 108 4.4 Restriction Analysis of pNB2.1 and pNBl 110 4.4.1 Introduction 110 4.4.2 Restriction Map of pNB2.1 111 4.4.3 A Restriction Map for pNBl 111 4.4.4 Discussion 111 4.5 Subcloning of pNBl - Selection of pTB8 115 4.5.1 Introduction 115 4.5.2 Subcloning of a 3.6 kb Pstl Fragment Containing valS into Ml 3 116 4.5.3 Discussion 118 CHAPTER 5. ANALYSIS OF THE CLONED vaLS GENE PRODUCT 5.1 Introduction 120 5.2 Thermostability of the Cloned B. stearothermophilus ValRS 121 5.2.1 Introduction 121 5.2.2 Results and Discussion 122 5.3 Analysis of Cloned ValRS by SDS— Polyacrylamide Gel Electrophoresis 124 5.4 Kinetic Measurements for Purified B. stearothermophilus ValRS 126 5.4.1 Introduction and Methods 126 5.4.2 Results and Discussion 127 5.5 Expression of valS Cloned into Ml 3 132 5.5.1 Introduction 132 5.5.2 Preliminary Aminoacylation Results 132 5.5.3 Orientation of Ml 3 valS Clones 133 5.5.4 The Class II valS Clones are Induced by IPTG 136 5.5.5 Discussion 140 5.6 Conclusions 143 CHAPTER 6. EXPERIMENTAL: DNA SEQUENCING 6.1 Introduction 147 Dideoxy Chain-termination Sequencing 147 The Use of Ml 3 Vectors in Chain- Termination Sequencing 148 Shotgun Cloning and Sequencing in M13 150 Sequencing of valS by the Chain- termination Method 151 Introduction 151 Materials 152 Bacterial Strains 152 Phage Vectors 152 Enzymes 154 Radiochemicals 154 Sequencing Primers 154 Nucleoside Triphosphates 155 Cloning of Sonicated DNA Fragments 157 Preparation of DNA Fragments 157 Sonication 157 Repair of Sheared Termini 158 Fractionation of 300-700 bp Fragments 158 Ligations 159 Preparation of Competent Cells 159 Transformation of Competent Cells 160 Preparation of Template DNA 160 Dideoxy Sequencing with [a-^^P] dATP 161 Separation of Radiolabelled Polynucleotides by Electrophoresis 162 Fixing and Autoradiography 164 Modifications for Sequencing with [a-35S] dATP 164 Modifications for Sequencing with dITP 165 Double-stranded Sequencing 166 Computer Software 166 7.