Gene Function

My aim in writing Gene Function has been to present an up-to-date picture ofthe molecular biology of Escherichia coli. I have not attempted a chronological description, believing that a mechanistic account is more useful for such a highly developed field. I have divided the book into four parts. Part I is a general introduction to bacterial systems, their genetic material, structure, composition and growth. It has seemed desirable to include herein a brief preview of the remaining text, to introduce the nomenclature and to help place subsequent chapters in perspective. The expression of genetic material and its perturbation through mutation is considered in Part II. Part III discusses how the transfer of prokaryotic genetic material can be mediated by plasmids and bacteriophages. It describes the DNA transactions involved (replication, recombination and repair) and ends with a description of the genetic and biochemical techniques employed in the study of gene organisation. Finally, Part IV considers the control of expression of bacterial, plasmid and phage genes. Key reviews are listed at the end of each chapter.

One: Introduction.- 1 The Bacterial Cell.- 1.1 The Nature of the Genetic Material.- 1.1.1 Structural Considerations.- (a) The Composition of Nucleic Acid.- (b) The Double Helix.- (c) Chromosome Structure.- 1.1.2 The Biological Role of Nucleic Acid: A Preview.- (a) Gene Expression.- (b) Replication.- (c) Genetic Recombination.- (d) DNA Repair.- (e) Symmetrical Recognition Sequences.- 1.1.3 Phenotype and Genotype.- 1.2 Cell Composition.- 1.2.1 The Cell Envelope.- 1.2.2 Cellular Appendages.- 1.3 Bacterial Growth.- 1.3.1 An Introduction to Metabolism.- (a) Production of the Chemical Intermediate, ATP.- (b) Biosynthetic Reactions.- (c) Pathway Regulation.- 1.3.2 Growth of Populations.- (a) Growth Requirements.- (b) Culture Characteristics.- (i) Lag Phase; (ii) Exponential Phase; (iii) Stationary Phase.- Two: Gene Expression.- 2 RNA and Protein Production.- 2.1 DNA Transcription.- 2.1.1 The Elements of Transcription.- (a) DNA-Dependent RNA Polymerase.- (b) Transcription Factors.- 2.1.2 The Mechanism of Transcription.- (a) The Transcription Cycle.- (i) Initiation; (ii) Elongation; (iii) Termination.- (b) Control: RNA Synthesis and Decay.- (c) Antibiotic Inhibitors of Transcription.- 2.2 RNA Translation.- 2.2.1 The Elements of Translation.- (a) The Genetic Code.- (b) Transfer RNA.- (c) The Ribosome.- 2.2.2 The Mechanism of Translation.- (a) The Ribosome Cycle.- (i) Initiation; (ii) Elongation; (iii) Termination.- (b) Post-Translational Modification.- (c) Control.- (d) Antibiotic Inhibitors of Translation.- 3 Mutation.- 3.1 Mutation Classification.- 3.1.1 Types of Point Mutation.- (a) Samesense Mutation.- (b) Missense Mutation.- (c) Nonsense Mutation.- (d) Frameshift Mutation.- 3.1.2 Conditional Mutants.- 3.2 Mutagenesis.- 3.2.1 The Molecular Basis of Mutagenesis.- (a) Spontaneous Mutation.- (b) Induced Mutation.- (i) Radiation; (ii) Chemical Mutagens.- (c) Mutational Hotspots.- 3.2.2 The Application of Mutagenesis to Bacterial Systems.- (a) Isolation of Independent Mutants that Carry Single Lesions.- (b) Mutant Selection.- (i) Replica-plating; (ii) Penicillin Enrichment.- 3.3 Suppression.- 3.3.1 Intragenic Suppression.- 3.3.2 Intergenic Suppression.- 3.3.3 Phenotypic Suppression: The Role of the Ribosome in Misreading.- 3.4 Summary: Mutation Identification.- Three: Gene Transfer.- 4 Plasmids.- 4.1 The F Plasmid.- 4.1.1 Vegetative Replication.- 4.1.2 F Transfer.- (a) The Mechanism of Transfer.- (b) Barriers to Transfer.- 4.1.3 F-Mediated Transfer.- (a) F integration: Hfr Formation.- (i) Insertion; (ii) Hfr Transfer; (iii) The One Hundred Minute Map.- (b) F Excision: F-Prime Formation.- 4.2 A Guide to Naturally-Occurring Plasmids.- 4.2.1 R Plasmids.- 4.2.2 Col Plasmids.- 5 Bacteriophages.- 5.1 Bacteriophages and their Life-Cycles.- 5.1.1 Anatomical Considerations.- (a) Phage Morphology.- (b) The Viral Genome.- 5.1.2 Phage Infection.- (a) The Lytic Response.- (b) Morphogenesis.- (c) The Lysogenic Response.- 5.1.3 Phage Methodology.- (a) Titration: The Plaque Assay.- (b) Production of Phage Lines.- 5.2 Bacteriophage-Mediated Gene Transfer.- 5.2.1 Generalised Transduction.- 5.2.2 Specialised Transduction.- 6 Reactions of DNA.- 6.1 DNA Replication.- 6.1.1 The Elements of Replication.- (a) DNA-Dependent DNA Polymerases.- (b) Protein Components of the Replication Fork.- 6.1.2 The Mechanism of Bacterial Replication.- (a) Bidirectional Replication of the E. coli Chromosome.- (i) Initiation; (ii) Elongation; (iii) Termination.- (b) The Role of the Cell Envelope in Replication.- (c) Control of DNA Replication.- (d) Antibiotic Inhibitors of Replication.- 6.1.3 Replication of Small Genetic Elements.- (a) Plasmid Production.- (b) Viral DNA Synthesis.- 6.2 Genetic Recombination.- 6.2.1 General Recombination.- (a) The Elements of General Recombination.- (i) The recA Gene Product; (ii) The Involvement of Other Gene Products in Recombination.- (b) Recombination Pathways.- (i) recA-mediated Pathways; (ii) The To Pathway.- (c) The Mechanism of General Recombination.- (i) Strand Breakage; (ii) Strand Pairing; (iii) Strand Invasion; (iv) Chiasma Formation; (v) Breakage and Reunion; (vi) Mismatch Repair.- 6.2.2 Non-Homologous Recombination.- (a) Site-Specific Recombination.- (i) Bacteriophages; (ii) Insertion Sequences and Transposons.- (b) Illegitimate Recombination.- 6.2.3 Phylogenetic Implications of Recombination.- 6.3 DNA Repair.- 6.3.1 The Elements of DNA Repair.- (a) Specific Repair Enzymes.- 6.3.2 The Mechanism of DNA Repair.- (a) Pre-Replication Repair.- (i) Excision Repair; (ii) Photoreactivation; (iii) Base Replacement.- (b) Post-Replication Repair.- (i) Daughter-strand Gap Repair; (ii) Error-prone Induced Repair.- 7 Investigation of Gene Structure and Function.- 7.1 Genetic Analysis of Bacteria and their Viruses.- 7.1.1 The Elements of Genetic Analysis.- (a) The cis-trans Complementation Test: The Gene as a Unit of Function.- (b) Recombination: An Indication of Genetic Distance.- 7.1.2 Mapping Bacterial Genes.- (a) Conjugation.- (i) Hfr Mating; (ii) F-Prime Plasmids.- (b) Transduction.- (i) P1 Generalised Transduction; (ii) Specialised Transduction.- (c) Mutagenesis by DNA Insertion Elements.- (i) Bacteriophages as Transposable Genetic Elements; (ii) Insertion Sequences and Transposons.- (d) Pathway Analysis.- 7.1.3 Mapping Phage Genes.- 7.2 Monitoring Gene Function.- 7.2.1 Protein Biosynthesis.- 7.2.2 RNA Production.- 7.3 Physical Analysis of Gene Structure.- 7.3.1 DNA Isolation.- 7.3.2 Heteroduplex Analysis.- 7.3.3 Restriction Technology.- (a) Restriction Cleavage Maps.- (b) DNA Cloning.- Four: Gene Regulation.- 8 Operon Control.- 8.1 Transcriptional Control.- 8.1.1 Control at RNA Chain Initiation.- (a) Promoter Control.- (i) The Elements of Promoter Control; (ii) Promoter Control Circuits.- (b) Cis- and Trans-Acting Regulatory Mutations.- 8.1.2 Control at RNA Chain Termination.- (a) Polarity and its Suppression.- (b) Attenuator Control.- 8.2 Post-Transcriptional Control.- 8.2.1 Control at the Level of the Transcript.- 8.2.2 Translational Control.- 9 Control of Bacterial Gene Expression.- 9.1 Catabolite-Controlled Operons: The Lactose System.- 9.1.1 The lac Operon.- 9.1.2 lac Promoter Control.- (a) Induction and Repression.- (b) Catabolite Control.- 9.2 Attenuator-Controlled Operons: The Tryptophan System.- 9.2.1 The trp Operon.- 9.2.2 trp Control.- (a) trp Promoter Control.- (b) trp Attenuator Control.- 9.3 Multivalent Control of Transcriptional-Translational Operons.- 9.3.1 Organisation of the Genes of the Transcription-Translation Apparatus.- (a) RNA Polymerase Operons.- (b) RNA Operons.- (i) Ribosomal RNA Operons; (ii) Transfer RNA Operons.- (c) Ribosomal Protein Operons.- 9.3.2 Control of Transcriptional-Translational Operons.- (a) Metabolic Control.- (b) Stringent Control.- (c) Co-Transcriptional and Post-Transcriptional Control.- 10 Control of Extrachromosomal Genetic Elements.- 10.1 Regulation of F Plasmid Transactions.- 10.1.1 Organisation of the F Plasmid.- (a) The Transfer Region.- (b) The Replication Region.- (c) The Insertion Region.- 10.1.2 Interaction between F and F-like R Plasmids.- 10.1.3 The ‘Life-Cycle’ of the F Plasmid.- 10.2 Regulation of Phage Lambda Development.- 10.2.1 Organisation of the Intracellular Viral Chromosome.- (a) The Regulatory Region.- (b) The Site-Specific Recombination Region.- (c) Replication Elements.- (d) The Morphogenetic Region.- 10.2.2 Lysogen Formation.- (a) Establishment of Lysogeny: PE-promoted Transcription.- (b) Maintenance of Lysogeny: PM-promoted Transcription.- 10.2.3 Prophage Induction.- (a) Repressor Inactivation.- (b) Immediate-Early Transcription: PL- and PR-promoted Events.- (c) Delayed-Early Transcription: PL and PR-promoted Readthrough Past the Early Terminators tL, tR, and tR2.- (d) Late Transcription: PR’-promoted Readthrough.- 10.2.4 The Lysogenic-Lytic ‘Decision’.