Maximizing Gene Expression (eBook)

Front Cover 1
Maximizing Gene Expression 6
Copyright Page 7
Table of Contents 10
Contributors 8
Preface 14
Chapter 1. E. Coli Promoters 16
1.1 Defining Promoters 17
1.2 Structure Analysis 23
1.3 Steps in Transcription Initiation 28
1.4 Structure-Function Correlation 33
1.5 Regulation of Transcription Initiation 37
1.6 Conclusion 44
References 44
Chapter 2. Yeast Promoters 50
2.1 Transcription in Yeast 52
2.2 Methods for Studying Yeast Promoters 53
2.3 Upstream Promoter Elements 58
2.4 The TATA Promoter Element 67
2.5 Elements that Select Initiation Sites 69
2.6 Transcription Regulation 71
2.7 Regulatory Proteins 75
2.8 Other Aspects of Regulation 79
2.9 Complex Promoter Organization 82
2.10 Molecular Mechanisms: Inferences and Speculations 86
References 90
Chapter 3. Protein Coding Genes of Higher Eukaryotes: Promoter Elements and trans-Acting Factors 94
3.1 The TATA Box and the Cap Site 95
3.2 The Upstream Promoter Elements 96
3.3 Enhancer Elements 98
3.4 Other trans-Acting Factors 103
3.5 Conclusion and Prospects 105
References 107
Chapter 4. The Instability of Messenger RNA in Bacteria 116
4.1 Some Fundamental Observations and Their Significance 118
4.2 Mechanistic Models 121
4.3 Search for Specific Enzymes for mRNA Degradation 130
4.4 The Search for Targets 132
4.5 Searching for New Ends 146
4.6 Searching for a Model 149
4.7 Conclusions 152
References 154
Chapter 5. Replication Control of the ColE1-Type Plasmids 158
5.1 Incompatibility 159
5.2 Replication of ColE1 DNA in Vitro 160
5.3 RNA I and Primer Processing 161
5.4 RNA I Secondary Structure 162
5.5 Mutations in RNA I and Primer That Define Domains of Interaction 165
5.6 Analysis of the RNA I-Primer Interaction 169
5.7 The Replication Primer 170
5.8 Replication-Defective Mutants 173
5.9 Temperature-Sensitive Replication Mutants 174
5.10 The rop Function 175
5.11 Partition and Stability Functions 178
5.12 Host Contributions to ColE1 Replication 180
5.13 On the Control of the Control Elements 180
5.14 Implications for Maximizing Gene Expression 181
References 183
Chapter 6. Copy Number and Stability of Yeast Plasmids 186
6.1 Vectors for DNA Cloning in Saccharomyces Cerevisiae 187
6.2 Elements of the 2µ Circle Replication System 192
6.3 Factors Affecting the Stability and Copy Number of Hybrid2µ-Based Plasmids 200
6.4 Conclusion 204
References 206
Chapter 7. Translation Initiation 210
7.1 Prokaryotes 211
7.2 Eukaryotes 228
7.3 Conclusion 234
References 235
Chapter 8. Biased Codon Usage: An Exploration of Its Role in Optimization of Translation 240
8.1 Codon Usage 240
8.2 Physiological Aspects of Codon Usage 263
8.3 Codon Context and tRNA-tRNA Interaction 276
8.4 Evolutionary Aspects of Codon Usage 286
8.5 Altering Codon Bias Experimentally 290
References 292
Chapter 9. The Selective Degradation of Abnormal Proteins in Bacteria 302
9.1 Proteins Rapidly Hydrolyzed in E. Coli 303
9.2 Intracellular Aggregates of Abnormal Polypeptides 306
9.3 The Energy Requirement and Pathway for Protein Breakdown 308
9.4 ATP-Stimulated Proteolysis in Cell-Free Extracts 311
9.5 Proteolytic Enzymes in E. Coli 312
9.6 Protease La, the ATP-Dependent Protease 314
9.7 The ATP-Dependent Proteolytic Mechanism 319
9.8 Regulation of Ion Expression 321
9.9 Protein Breakdown and the Heat Shock Response 322
9.10 Unanswered Questions 326
References 326
Chapter 10. Detection of Proteins Produced by Recombinant DNA Techniques 330
10.1 Complementation of E. Coli Mutants 331
10.2 Complementation of Yeast Mutants 334
10.3 Complementation in Mammalian Cells 335
10.4 Rescue of the Sequence Conferring the New Phenotype 337
10.5 Complementation in Drosophila 343
10.6 Detection Using Immunological Methods 344
10.7 Detection Using Antisera Raised Against Synthetic Peptides 351
References 353
Chapter 11. Mechanism and Practice 360
11.1 Transcription Initiation 361
11.2 Translation Initiation 364
11.3 Messenger Stability and Secondary Structure 366
11.4 Gene Amplification 369
11.5 Protein Stability 373
References 376
Index 380