WIXLIB

xii ContentsProblemsReferences12 Induction, Repression, and the araBAD OperonThe Sugar Arabinose and Arabinose MetabolismGenetics of the Arabinose SystemDetection and Isolation of AraC ProteinRepression by AraCRegulating AraC SynthesisBinding Sites of the ara Regulatory ProteinsDNA Looping and Repression of araBADIn vivo Footprinting Demonstration of LoopingHow AraC Protein Loops and UnloopsWhy Looping is Biologically SensibleWhy Positive Regulators are a Good IdeaProblemsReferences13 Attenuation and the trp OperonThe Aromatic Amino Acid Synthetic Pathway and its RegulationRapid Induction Capabilities of the trp OperonThe Serendipitous Discovery of trp Enzyme HypersynthesisEarly Explorations of the Hypersynthesistrp Multiple Secondary Structures in trp Leader RNACoupling Translation to TerminationRNA Secondary Structure and the Attenuation MechanismOther Attenuated Systems: Operons, Bacillus subtilis and HIVProblemsReferences14 Lambda Phage Genes and Regulatory 6377379385386388390392396397399400402404409A. The Structure and Biology of Lambda410The Physical Structure of LambdaThe Genetic Structure of LambdaLysogeny and ImmunityLambda’s Relatives and Lambda Hybrids410411413414B. Chronology of a Lytic Infective Cycle415Lambda Adsorption to CellsEarly Transcription of Genes N and CroN Protein and Antitermination of Early Gene TranscriptionThe Role of Cro ProteinInitiating DNA Synthesis with the O and P ProteinsProteins Kil, γ, β, and ExoQ Protein and Late Proten SynthesisLysis415416417418418419420421

Contents xiiiC. The Lysogenic Infective Cycle and Induction of Lysogens422Chronology of Becoming a LysogenSite for Cro Repression and CI ActivationCooperativity in Repressor Binding and its MeasurementThe Need for and the Realization of Hair-Trigger InductionInduction from the Lysogenic StateEntropy, a Basis for Lambda Repressor 343515 Xenopus 5S RNA SynthesisBiology of 5S RNA Synthesis in XenopusIn vitro 5S RNA SynthesisTFIIIA Binding to the Middle of its Gene as Well as to RNASwitching from Oocyte to Somatic 5S SynthesisStructure and Function of TFIIIAProblemsReferences16 Regulation of Mating Type in YeastThe Yeast Cell CycleMating Type Conversion in Saccharomyces cerevisiaeCloning the Mating Type Loci in YeastTransfer of Mating Type Gene Copies to an Expression SiteStructure of the Mating Type LociThe Expression and Recombination ParadoxesSilencing HML and HMRIsolation of α2 Proteinα2 and MCM1Sterile Mutants, Membrane Receptors and G FactorsDNA Cleavage at the MAT LocusDNA Strand Inheritance and Switching in Fission YeastProblemsReferences17 Genes Regulating DevelopmentGeneral Considerations on SignalingOutline of Early Drosophila DevelopmentClassical EmbryologyUsing Genetics to Begin Study of Developmental SystemsCloning Developmental GenesEnhancer Traps for Detecting and Cloning Developmental GenesExpression Patterns of Developmental GenesSimilarities Among Developmental GenesOverall Model of Drosophila Early 7488491491

xiv ContentsProblemsReferences18 Lambda Phage Integration and ExcisionMapping Integrated LambdaSimultaneous Deletion of Chromosomal and Lambda DNADNA Heteroduplexes Prove that Lambda IntegratesGene Order Permutation and the Campbell ModelIsolation of Integration-Defective MutantsIsolation of Excision-Deficient MutantsProperties of the int and xis Gene ProductsIncorrect Excision and gal and bio Transducing PhageTransducing Phage Carrying Genes Other than gal and bioUse of Transducing Phage to Study Integration and ExcisionThe Double att Phage, att2Demonstrating Xis is UnstableInhibition By a Downstream ElementIn vitro Assay of Integration and ExcisionHost Proteins Involved in Integration and ExcisionStructure of the att RegionsStructure of the IntasomeHolliday Structures and Branch Migration in IntegrationProblemsReferences19 Transposable Genetic ElementsIS Elements in BacteriaStructure and Properties of IS ElementsDiscovery of Tn ElementsStructure and Properties of Tn ElementsInverting DNA Segments by Recombination, Flagellin SynthesisMu Phage As a Giant Transposable ElementAn Invertible Segment of Mu PhageIn vitro Transposition, Threading or Global Topology?Hopping by Tn10Retrotransposons in Higher CellsAn RNA Transposition IntermediateP Elements and TransformationP Element Hopping by Chromosome RescueProblemsReferences20 Generating Genetic Diversity: AntibodiesThe Basic Adaptive Immune ResponseTelling the Difference Between Foreign and SelfThe Number of Different Antibodies 4545547550552553555557558563563565566

Contents xvMyelomas and Monoclonal AntibodiesThe Structure of AntibodiesMany Copies of V Genes and Only a Few C GenesThe J RegionsThe D Regions in H ChainsInduced Mutations and Antibody DiversityClass Switching of Heavy ChainsEnhancers and Expression of Immunoglobulin GenesThe AIDS VirusEngineering Antibody Synthesis in BacteriaAssaying for Sequence Requirements of Gene RearrangementsCloning the RecombinaseProblemsReferences21 Biological Assembly, Ribosomes and Lambda A. Ribosome Assembly592RNAse and RibosomesThe Global Structure of RibosomesAssembly of RibosomesExperiments with in vitro Ribosome AssemblyDetermining Details of Local Ribosomal Structure592593595597599B. Lambda Phage Assembly601General AspectsThe Geometry of CapsidsThe Structure of the Lambda ParticleThe Head Assembly Sequence and Host ProteinsPackaging the DNA and Formation of the cos EndsFormation of the TailIn vitro 061322 ChemotaxisAssaying ChemotaxisFundamental Properties of ChemotaxisGenetics of Motility and ChemotaxisHow Cells SwimThe Mechanism of ChemotaxisThe Energy for ChemotaxisAdaptationMethylation and AdaptationPhosphorylation and the Rapid 632633635637

xvi Contents23 Oncogenesis, Molecular AspectsBacterially Induced Tumors in PlantsTransformation and Oncogenesis by Damaging the ChromosomeIdentifying a Nucleotide Change Causing CancerRetroviruses and CancerCellular Counterparts of Retroviral OncogenesIdentification of the src and sis Gene ProductsDNA Tumor VirusesRecessive Oncogenic Mutations, Tumor SuppressorsThe ras-fos-jun PathwayDirections for Future Research in Molecular 58660661661663Hints and Solutions to Odd-Numbered Problems667Index685

An Overview of CellStructure and Function1In this book we will be concerned with the basics of the macromolecularinteractions that affect cellular processes. The basic tools for suchstudies are genetics, chemistry, and physics. For the most part, we willbe concerned with understanding processes that occur within cells, suchas DNA synthesis, protein synthesis, and regulation of gene activity. Theinitial studies of these processes utilize whole cells. These normally arefollowed by deeper biochemical and biophysical studies of individualcomponents. Before beginning the main topics we should take time foran overview of cell structure and function. At the same time we shoulddevelop our intuitions about the time and distance scales relevant to themolecules and cells we will study.Many of the experiments discussed in this book were done with thebacterium Escherichia coli, the yeast Saccharomyces cerevisiae, and thefruit fly Drosophila melanogaster. Each of these organisms possessesunique characteristics making it particularly suitable for study. In fact,most of the research in molecular biology has been confined to thesethree organisms. The earliest and most extensive work has been donewith Escherichia coli. The growth of this oranism is rapid and inexpensive, and many of the most fundamental problems in biology aredisplayed by systems utilized by this bacterium. These problems aretherefore most efficiently studied there. The eukaryotic organisms arenecessary for study of phenomena not observed in bacteria, but parallelstudies on other bacteria and higher cells have revealed that the basicprinciples of cell operation are the same for all cell types.1

2 An Overview of Cell Structure and FunctionCell’s Need for Immense Amounts of InformationCells face enormous problems in growing. We can develop some idea ofthe situation by considering a totally self-sufficient toolmaking shop. Ifwe provide the shop with coal for energy and crude ores, analogous toa cell’s nutrient medium, then a very large collection of machines andtools is necessary merely to manufacture each of the parts present inthe shop. Still greater complexity would be added if we required thatthe shop be totally self-regulating and that each machine be self-assembling. Cells face and solve these types of problems. In addition, each ofthe chemical reactions necessary for growth of cells is carried out in anaqueous environment at near neutral pH. These are conditions thatwould cripple ordinary chemists.By the tool shop analogy, we expect cells to utilize large numbers of“parts,” and, also by analogy to factories, we expect each of these partsto be generated by a specialized machine devoted to production of justone type of part. Indeed, biochemists’ studies of metabolic pathwayshave revealed that an E. coli cell contains about 1,000 types of parts, orsmall molecules, and that each is generated by a specialized machine,an enzyme. The information required to specify the structure of evenone machine is immense, a fact made apparent by trying to describe anobject without pictures and drawings. Thus, it is reasonable, and indeedit has been found that cells function with truly immense amounts ofinformation.DNA is the cell’s library in which information is stored in its sequenceof nucleotides. Evolution has built into this library the informationnecessary for cells’ growth and division. Because of the great value ofthe DNA library, it is natural that it be carefully protected and preserved.Except for some of the simplest viruses, cells keep duplicates of theinformation by using a pair of self-complementary DNA strands. Eachstrand contains a complete copy of the information, and chemical orphysical damage to one strand is recognized by special enzymes and isrepaired by making use of information contained on the oppositestrand. More complex cells further preserve their information by possessing duplicate DNA duplexes.Much of the recent activity in molecular biology can be understoodin terms of the cell’s library. This library contains the informationnecessary to construct the different cellular machines. Clearly, such alibrary contains far too much information for the cell to use at any onetime. Therefore mechanisms have developed to recognize the need forparticular portions, “books,” of the information and read this out of thelibrary in the form of usable copies. In cellular terms, this is theregulation of gene activity.Rudiments of Prokaryotic Cell StructureA typical prokaryote, E. coli, is a rod capped with hemispheres (Fig. 1.1).It is 1–3 µ (10-4 cm 1 µ 104 Å) long and 0.75 µ in diameter. Such a

Rudiments of Prokaryotic Cell Structure 3RibosomesNuclearregionCellenvelope0.75µFigure 1.1 The dimensions of atypical E. coli cell.2µcell contains about 2 10-13 g of protein, 2 10-14 g of RNA that is mostlyribosomal RNA, and 6 10-15 g of DNA.The cell envelope consists of three parts, an inner and outer membrane and an intervening peptidoglycan layer (Fig. 1.2). The outersurface of the outer membrane is largely lipopolysaccharides. These areattached to lipids in the outer half of the outer membrane. The polysaccharides protect the outer membrane from detergent-like moleculesfound in our digestive tract.outer membrane The outer membrane alsoconsists of matrix proteins that form pores small enough to exclude thedetergent-like bile salts, but large enough to permit passage of smallmolecules and phospholipids.Figure 1.2 Schematic drawing of the structure of the envelope of an E. nLipidsOutermembranePeptidoglycanor cell wallPeriplasmicspaceInnermembranePeriplasmic proteinPhospholipidsProteinsPhospholipid

4 An Overview of Cell Structure and 0-60 units in E. coliMN-acetylmuramic acidCH2 OHCH2 OHOH HOH H HOHHNHCCH 3OH HOOHHOHHOCCOOHCH3NHCOCH3(b)Figure 1.3 Structure of the cell wall showing the alternating N-acetylglucosamine N-acetylmuramic acid units. Each N-acetylmuramic acid possessesa peptide, but only a few are crosslinked in E. coli.The major shape-determining factor of cells is the peptidoglycanlayer or cell wall (Fig. 1.3). It lies beneath the outer membrane and is asingle molecule containing many polysaccharide chains crosslinked byshort peptides (Fig. 1.4). The outer membrane is attached to the peptidoglycan layer by about 106 lipoprotein molecules. The protein end ofeach of these is covalently attached to the diaminopimelic acid in thepeptidoglycan. The lipid end is buried in the outer membrane.The innermost of the three cell envelope layers is the inner orcytoplasmic membrane. It consists of many proteins embedded in aphospholipid bilayer. The space between the inner membrane and theouter membrane that contains the peptidoglycan layer is known as theperiplasmic space. The cell wall and membranes contain about 20% ofthe cellular protein. After cell disruption by sonicating or grinding, mostof this protein is still contained in fragments of wall and membrane andcan be easily pelleted by low-speed centrifugation.The cytoplasm within the inner membrane is a protein solution atabout 200 mg/ml, about 20 times more concentrated than the usualcell-free extracts used in the laboratory. Some proteins in the cytoplasmmay constitute as little as 0.0001% by weight of the total cellular proteinwhereas others may be found at levels as high as 5%. In terms ofconcentrations, this is from 10-8 M to 2 10-4 M, and in a bacterial cellthis is from 10 to 200,000 molecules per cell. The concentrations ofmany of the proteins vary with growth conditions, and a current research area is the study of the cellular mechanisms responsible for thevariations.The majority of the more than 2,000 different types of proteins foundwithin a bacterial cell are located in the cytoplasm. One question yet to

Rudiments of Prokaryotic Cell Structure 5N-acetylmuramic acidOHOOCCHNHAcOOHHOOHOH CCH 3 CCNCCNHCH 3L-AlaCHD-GluN-acetylmuramic acidCH2(CH 2) 3CH 2OHCNCOHCH 3CNCHOHOCHmeso -DAPD-AlaN-acetylmuramic Figure 1.4 Structure of the peptide crosslinking N-acetylmuramic acid units.DAP is diaminopimelic acid.be answered about these proteins is how they manage to exist in the cellwithout adhering to each other and forming aggregates since polypeptides can easily bind to each other. Frequently when a bacterium isengineered for the over-synthesis of a foreign protein, amorphousprecipitates called inclusion bodies form in the cytoplasm. Sometimesthese result from delayed folding of the new protein, and occasionallythey are the result of chance coprecipitation of a bacterial protein andthe newly introduced protein. Similarly, one might also expect anoccasional mutation to inactivate simultaneously two apparently unrelated proteins by the coprecipitation of the mutated protein and someother protein into an inactive aggregate, and occasionally this doesoccur.The cell’s DNA and about 10,000 ribosomes also reside in the cytoplasm. The ribosomes consist of about one-third protein and two-thirdsRNA and are roughly spherical with a diameter of about 200 Å. The DNAin the cytoplasm is not surrounded by a nuclear membrane as it is inthe cells of higher organisms, but nonetheless it is usually confined toa portion of the cellular interior. In electron micrographs of cells, thehighly compacted DNA can be seen as a stringy mass occupying aboutone tenth of the interior volume, and the ribosomes appear as granulesuniformly scattered through the cytoplasm.

6 An Overview of Cell Structure and FunctionRudiments of Eukaryotic Cell StructureA typical eukaryotic cell is 10 µ in diameter, making its volume about1,000 times that of a bacterial cell. Like bacteria, eukaryotic cells containcell membranes, cytoplasmic proteins, DNA, and ribosomes, albeit ofsomewhat different structure from the corresponding prokaryotic elements (Fig. 1.5). Eukaryotic cells, however, possess many structuralfeatures that even more clearly distinguish them from prokaryotic cells.Within the eukaryotic cytoplasm are a number of structural proteinsthat form networks. Microtubules, actin, intermediate filaments, andthin filaments form four main categories of fibers found within eukaryotic cells. Fibers within the cell provide a rigid structural skeleton,participate in vesicle and chromosome movement, and participate inchanging the cell shape so that it can move. They also bind the majorityof the ribosomes.The DNA of eukaryotic cells does not freely mix with the cytoplasm,but is confined within a nuclear membrane. Normally only small proteins of molecular weight less than 20 to 40,000 can freely enter thenucleus through the nuclear membrane. Larger proteins and nuclearRNAs enter the nucleus through special nuclear pores. These are largestructures that actively transport proteins or RNAs into or out of thenucleus. In each cell cycle, the nuclear membrane dissociates, and thenlater reaggregates. The DNA itself is tightly complexed with a class ofproteins called histones, whose main function appears to be to help DNAretain a condensed state. When the cell divides, a special apparatuscalled the spindle, and consisting in part of microtubules, is necessaryto pull the chromosomes into the daughter cells.Eukaryotic cells also contain specialized organelles such as mitochondria, which perform oxidative phosphorylation to generate thecell’s needed chemical energy. In many respects mitochondria resemblebacteria and, in fact, appear to have evolved from bacteria. They containDNA, usually in the form of a circular chromosome like that of E. coliFigure 1.5 S c h e m a t i cdrawing of a µNuclearmembraneNucleusEndoplasmicreticulumGolgi ap

Printed in the United States of America on acid-free paper The Johns Hopkins University Press 2715 North Charles Street Baltimore, Maryland 21218-4319 The Johns Hopkins Press Ltd., London Library of Congress Cataloging-in-Publication Data Schleif, Robert F. Genetics and molec