Biochemistry andMolecular Biology
William H. ElliottDepartment of Molecular Biosciences, University of Adelaide
Daphne C. ElliottSchool of Biological Sciences, Flinders University of South Australia
OXFORDUNIVERSITY PRESS
Contents
List of abbreviations XXV
Part 1 Introduction to thechemical reactions of the cell
Chapter 1 Chemistry, energy, and metabolism
• What determines whether a chemical reaction is possible?
• Reversible and irreversible reactions and AG values
• The importance of irreversible reactions in the strategy ofmetabolism
• Why is this metabolic strategy used in the cell?
• How are AG values obtained?
• Standard free energy values and equilibrium constants
• Given that a reaction has a negative A6 value, what
determines whether it actually takes place at a
perceptible rate in the cell?
How is food breakdown in cells coupled to driveenergy-requiring reactions in the cell?
• The high-energy phosphate compound
What is a 'high-energy phosphate compound'?
• What are the structural features of high-energyphosphate compounds?
• What transports the—©around the cell?
• How does ATP perform chemical work?
• How does ATP drive other types of work?
• A note on the relationship between AMP, ADP, and ATP
Weak bonds and free-energy changes
• What causes weak bond formation and breakage?
• The vital role of weak bonds in molecular recognition
Appendix: Buffers and pKa values
Further reading
Problems
Chapter 2 Enzymes
Enzyme catalysis
• The nature of enzyme catalysis
• The induced fit mechanism of enzyme catalysis
Enzyme kinetics
<• Hyperbolic kinetics of a 'classical' enzyme
• Allosteric enzymes
General properties of enzymes
• Nomenclature of enzymes
» Isozymes
o Enzyme cofactors and activators
• Effect of pH on enzymes
• Effect of temperature on enzymes
• Effect of inhibitors on enzymes
Reversible inhibitors
Competitive and noncompetitive inhibitors
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Further reading
Problems
WKPSM Part 2 Structure of proteins• p S S f and membranes
Chapter 3 The structure of proteins
The primary structure of proteins
• What is a native protein?
• What are the basic considerations which determine thethree-dimensional structure of a protein?
• Structures of the 20 amino acids
Hydrophobic amino acids
Hydrophilic amino acids
Amino acids for special purposes
lonization of amino acidsSymbols for amino acids
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Xiv CONTENTS
The different levels of protein structure—primary,
secondary, tertiary, and quaternary 38
• Secondary structure of proteins 39
The a helix 39The p-pleated sheet 40
Connecting loops 41
• Tertiary structure of proteins 41
How are proteins made up from the three motifs—the a helix, the
^-pleated sheet, and connecting loops? 41
What forces hold the tertiary structure in position? 42
Where do the disulfide or S-S covalent bonds come into proteinstructure? 42
• Quaternary structure of proteins 44
• Membrane proteins 44
• Conjugated proteins 44
• What are protein modules or domains? 45
Why should domains be of interest? 45
• The problem of protein folding 46
What are the structural features of enzyme proteins thatconfer catalytic activity on them? 47
• A special note 47
• Mechanism of the chymotrypsin reaction 47
• The catalytic triad of the active centre 48
• The reactions at the catalytic centre of chymotrypsin 49
• What is the function of the aspartate residue of thecatalytic triad? 49
• Other serine proteases 51
• A brief description of other types of proteases 51
Extracellular matrix proteins 51
• Structure of collagens 52
Genetic diseases of collagen 54
• Structure of elastin 54
• Structure of proteoglycans 54
• Adhesion proteins of the extracellular matrix 56
Experimental handling and separation of proteins 57
• Column chromatography 57
• SDS polyacrylamide gel electrophoresis 58
Methods of protein sequencing 60
• Deduction of a protein sequence from the nucleotide
structure of its gene 61
Protein databases and proteomics 61
• A note on where we are at this point in the book 62
Further reading 62
Problems 63
Chapter 4 The cell membrane: a structuredepending only on weak forces 67
An overview of cellular membranes 67
Why are cell membranes needed? 68
The lipid bilayer 70
• What are the polar lipid constituents of cell membranes? 70
• What are the polar groups attached to the phosphatidic
acid? 71
« A note on membrane lipid nomenclature 74
a Why are there so many different types of membrane
lipids? 74
• What are the fatty acid components of membrane lipids? 75
• What is cholesterol doing in membranes? 75
• The self-sealing character of the lipid bilayer 76
• Permeability characteristics of the lipid bilayer 76
Membrane proteins and membrane design 77
What holds integral proteins in the lipid bilayer? 77
• Anchoring of peripheral membrane proteins to membranes 78
• Glycoproteins or membrane proteins with sugars attachedon the exterior surface 79
Functions of membranes 80
• Transport of substances in and out of the cell 80
Active transport 80
Mechanism of the Na*/K* pump 80
Symport systems 82
Antiport systems 82
Uniport systems 82
Passive transport or facilitated diffusion 82
• Gated ion channels 83
• Nerve impulse transmission 83
The acetylcholine-gated Na*/K* channel or acetylcholine receptor 84
How does acetylcholine binding to a membrane receptor result in a
nerve impulse? 84
How is the initial signal propagated along nerve axons? 86
Mechanism of control of the voltage-gated Na* and K* channels 88
Why doesn't the Na+/K* pump conflict with the propagation of actionpotentials? 89
• Signal transduction—a brief preliminary note 89
• Role of the cell membrane in maintaining the shape of thecell and in cell mobility 90
• Cell-cell interactions—tight junctions and gap junctionsand cellular adhesive proteins 90
Further reading 91
Problems 92
CONTENTS XV
Part 3
Chapter 5 Digestion a n d absorption of food 97
Chemistry of foodstuffs 97
Digestion and absorption 97
• Anatomy of the digestive tract 97
• What are the energy considerations in digestion andabsorption? 98
• A major problem in digestion—why doesn't the bodydigest itself? 98
Zymogen or proenzyme production 99
Protection of intestinal epithelial cells by mucus 99
• Digestion of proteins 99
• HCI production in the stomach 99
• Pepsin, the proteolytic enzyme of the stomach 99
• Completion of protein digestion in the small intestine 100
• Activation of the pancreatic proenzymes 100
• Absorption of amino acids into the bloodstream 101
• Digestion of carbohydrates 101
• Digestion of starch 101
• Digestion and absorption of fat 103
• What happens to the fatty acids and monoacylglycerol
absorbed into the intestinal brush border cells? 104
Resynthesis of neutral fat in the absorptive cell 104
• What are chylomicrons? 105• Digestion of other components of food 106
Further reading 106
Problems 106
Chapter 6 Preliminary outline of fueldistribution and utilization by the differenttissues of the body
Purposes of metabolismStorage of rood in the body
• How are the different foods stored in cells?
Glucose storage as glycogen
Storage of fat in the body
Are amino acids stored by the body?
Characteristics of different tissues in terms of energymetabolism
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Overall control of the logistics of food distribution in thebody by hormones 112
o Post-absorptive phase 112
o Fasting condition 113
° Prolonged fasting or starvation 113
o The situation in diabetes 113
o The emergency situation—fight or flight 113
Problems 114
Chapter 7 Biochemical mechanisms involved infood transport, storage, and mobilization 117
Glucose traffic in the body 117
o Mechanism of glycogen synthesis 117
° How is energy injected into the process? 118
o How does the liver release glucose? 120
o Why does liver have glucokinase and the other tissueshexokinase? 122
o What happens to other sugars absorbed from the
intestine? 122
Amino acid traffic in the body (in terms of fuel logistics) 124
Fat and cholesterol traffic in the body 125
o Uptake of fat from chylomicrons into cells 125
Logistics of fat and cholesterol movement in the body 125
o An overview 125o Utilization of cholesterol in the body 126
o Lipoproteins involved in fat and cholesterol movement in
the body 127
o Apolipoproteins 127
o Mechanism of TAG and cholesterol transport from the
liver and the reverse cholesterol transport in the body 127
How do cells get rid of cholesterol? 127
How does cholesterol exit cells to be picked up by HDL? 129
Cholesterol homeostasis in cells 130
Inhibitors of cholesterol synthesis 130
o How is fat released from adipose cells? 130
o How are free fatty acids carried in the blood? 131
Further reading 132
Problems 132
Chapter 8 Energy release fromn foodstuffs—apreliminary overview 135
Energy release from glucose 135
° The main phases of glucose oxidation 135
XVi CONTENTS
• Biological oxidation and hydrogen transfer systems
NAD*—an important electron carrier
FAD—another important electron carrier
• FAD, FMN, and their reduction
• Stages in the release of energy from glucose—firstglycolysis
• Stage 2 of glucose oxidation—the citric acid cycle
How is pyruvate fed into the citric acid cycle?
What is coenzyme A?
Oxidative decarboxylation of pyruvate
• Stage 3 of glucose oxidation—electron transport tooxygen
• The electron transport chain—a hierarchy of electroncarriers
Energy generation from oxidation of fat and amino acids
The interconvertibility of fuels
A survey of vitamins
Further reading
Problems
Chapter 9 Glycolysis, the citric acid cycle, andthe electron transport system: reactionsinvolved in these pathways
Stage 1—glycolysis
• Glucose or glycogen?
Why use ATP here at the beginning of glycolysis?
• Why is glucose-6-phosphate converted to fructose-6-phosphate?
• A note on the AG0' and AG values for the aldolase reaction
• Interconversion of dihydroxyacetone phosphate andglyceraldehyde-3-phosphate
• Gyceraldehyde-3-phosphate dehydrogenase—a stepgenerating a high-energy-phosphoryl compound
• The final steps in glycolysis
• The ATP balance sheet from glycolysis
• Reoxidation of cytoplasmic NADH by electron shuttlesystems
• Transport of pyruvate into the mitochondria
Stage 2—the citric acid cycle
• Conversion of pyruvate to acetyl-CoA-a preliminary stepbefore the cycle
• Components involved in the pyruvate dehydrogenasereaction
• The citric acid cycle as a water-splitting machine
• A simplified version of the citric acid cycle
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• Mechanisms of the citric acid cycle reactions 157
The synthesis of citrate 157
Conversion of citrate to a-ketoglutarate 157
• The C4 part of the cycle 158
Generation ofGTP coupled to splitting of succinyl-CoA 159Conversion ofsuccinate to oxaloacetate 159
• What determines the direction of the citric acid cycle? 160
• Stoichiometry of the cycle 160
• Topping up the citric acid cycle 161
Stage 3—the electron transport chain that conveyselectrons from NADH and FADH2 to oxygen 162
• The electron transport chain 162
Where does it take place? 162
Nature of the electron carriers in the chain 162
Arrangement of the electron carriers 163
• How is the free energy released by electron transport
used to form ATP? 164
• How are protons ejected? 166
• How does the proton gradient drive ATP synthesis?-ATP synthase 167
Components of the F, unit and their roles in the conversion ofADP
+ P,toATP 169
Activities of the enzyme catalytic centres on the F, subunit 169
How is the energy of proton flow through the Fo utilized for ATP
synthesis by the F,? 170
What is the role of the Fo unit ? 170
• Transport of ADP into mitochondria and ATP out 172
• The balance sheet of ATP production by electrontransport 173
• Yield of ATP from the oxidation of a molecule of glucoseto CO2 and H20 173
• Is ATP production the only use that is made of the
potential energy in the proton-motive force? 174
Further reading 174
Problems 175
Chapter 10 Energy release from fat 179
Mechanism of acetyl-CoA formation from fatty acids 180
• 'Activation' of fatty acids by formation of fatty acyl-CoAderivatives 180
• Transport of fatty acyl-CoA derivatives into mitochondria 180
• Conversion of fatty acyl-CoA to acetyl-CoA moleculesinside the mitochondrion 181
• Energy yield from fatty acid oxidation 181
CONTENTS xvii
Oxidation of unsaturated fat
Is the acetyl-CoA derived from fat breakdown alwaysdirectly fed into the citric acid cycle?
• How is acetoacetate made from acetyl-CoA?
Oxidation of odd-numbered carbon chain fatty acids
• Peroxisomal oxidation of fatty acids
Further reading
Problems
Chapter 11 A switch from catabolic toanabolic metabolism—first, the synthesis of fatand related compounds in the body
Mechanism of fat synthesis
• General principles of the process
• The acyl carrier protein (ACP) and the /3-ketoacyl synthase
• Mechanism of fatty acyl-CoA synthesis
• Organization of the fatty acid synthesis process
• The reductive steps in fatty acid synthesis
WhatisNADP*?
• Where does fatty acid synthesis take place?
Synthesis of unsaturated fatty acids
Synthesis of triacylglycerol and membrane lipids fromfatty acids
Synthesis of glycerophospholipids
• Site of membrane lipid synthesis
Synthesis of prostaglandins and related compounds
• The prostaglandins and thromboxanes
• Leukotrienes
• Synthesis of cholesterol
Further reading
Problems
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Chapter 12 Synthesis of glucose(gluconeogenesis)
• Mechanism of glucose synthesis from pyruvate
• What are the sources of pyruvate used by the liver forgluconeogenesis?
• Effects of ethanol metabolism on gluconeogenesis
Effects of ethanol metabolism on the NAD/NADH ratio in the liver cell
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• Synthesis of glucose from glycerol 204
• Synthesis of glucose via the glyoxylate cycle 204
Further reading 205
Problems 206
Chapter 13 Strategies for metabolic controland their application to carbohydrate andfat metabolism 209
Why are controls necessary? 209
» The potential danger of futile cycles in metabolism 210
How are enzyme activities controlled? 211
• Metabolic control by varying the amounts of enzymes 211
• Metabolic control by regulation of the activities ofenzymes in the cell 211
• Which enzymes in metabolic pathways are regulated? 211
• The nature of regulatory enzymes 212
Allosteric control of enzymes 212
• The mechanism of allosteric control of enzymes 212
What causes the sigmoidal response of reaction velocity tosubstrate concentration? 213
• Reversibility of allosteric control 214
« Allosteric control is a tremendously powerful metabolic
concept 214
Control of enzyme activity by phosphorylation 215
• The two classes of controls—intracellular mechanisms
and those dependent on extracellular signals 215
Hormonal control of metabolism 216
« How do glucagon, epinephrine, and insulin, work? 216• What is a second messenger? 216• What is the second messenger for glucagon and
epinephrine? 217
Control of carbohydrate metabolism 218
• Control of glucose uptake into cells 218
• Control of glycogen metabolism 219
• Activation of muscle phosphorylase by cAMP 220
• Reversal of phosphorylase activation 221
• Control of glycogen breakdown in the liver 222
• Control of glycogen synthase 222
• How does insulin inactivate GSK3? 223
• Control of glycolysis and gluconeogenesis 223
Allosteric controls 223
Hormonal control of glycolysis and gluconeogenesis 224
How does cAMP control PFK? 225
xviii CONTENTS
• Control of gluconeogenesis
• Control of pyruvate dehydrogenase, the citric acid cycle,and oxidative phosphorylation
• Regulation of the cellular ATP level by AMP-activatedprotein kinase
• Controls of fatty acid oxidation and synthesis
Nonhormonal controls
Hormonal controls on fat metabolism
• Insulin and diabetes
• A concluding note: metabolic control analysis
Further reading
Problems
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Problems227
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Chapter 14 Why should there be an alternativepathway of glucose oxidation—the pentosephosphate pathway? 235
• The oxidative steps 235
• The nonoxidative section and its purpose 235
• Reactions involved in the conversion of ribose-5-phosphate to glucose-6-phosphate 237
• Where does the complete oxidation of glucose come intoall of this? 238
• Why do red blood cells have the pentose phosphatepathway? 238
Further reading
Problems
Chapter 15 Raising electrons of water back upthe energy scale—photosynthesis
• Overview
• Site of photosynthesis—the chloroplast
• The photosynthetic apparatus and its organization in thethylakoid membrane
• What is chlorophyll?
• Mechanism of light-dependent reduction of NADP+
The water-splitting centre ofPSII
• How is ATP generated?
An explanatory note
• How is C02 converted to carbohydrate?
Where does the 3-phosphoglycerate come from?
Where does the ribulose-l:5-bisphosphate come from?
• Has evolution slipped up a bit?
• The C4 pathway
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Chapter 16 Amino acid metabolism 253
Nitrogen balance of the body 254
General metabolism of amino acids 254
• Aspects of amino acid metabolism 254
• Deamination of amino acids 255
Mechanism oftransamination reactions 256
Special deamination mechanisms 256
• Fate of the keto acid or carbon skeletons of deaminated
amino acids 257
• Phenylalanine metabolism has special interest 258
• Methionine and transfer of methyl groups 258
Where are the methyl groups transferred to? 259
Synthesis of amino acids 259
• Synthesis of glutamic acid 259
• Synthesis of aspartic acid and alanine 259
• Synthesis of serine 259
• Synthesis of glycine 259
Synthesis of other molecules from amino acids 260
What happens to the amino groups when they areremoved from amino acids?—the urea cycle 261
• Mechanism of arginine synthesis 262
• Conversion of citrulline to arginine 262
• How is the amino nitrogen transported from extrahepatictissues to the liver to be converted into urea? 263
Transport of ammonia in the blood as glutamine 263
Transport of amino nitrogen in the blood as alanine 263
Further reading 264
Problems 264
Chapter 17 Cellular disposal of unwantedmolecules
Lysosomes
• Mechanism of receptor-mediated endocytosis
• Lysosomal storage diseases
Peroxisomes
Proteasomes
• What are the problems to be solved by the cell toselectively destroy proteins?
• The structure of proteasomes
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CONTENTS Xix
• Selection of proteins for destruction by proteasomes—theubiquitination system 272
• What determines which proteins are ubiquitinated? 273
• The role of proteasomes in the immune system 273
Further reading 273
Problems 274
Chapter 18 Enzymic protective mechanisms inthe body
Blood clotting
• What are the signals that clot formation is needed?
• How does thrombin cause thrombus (clot) formation?
• Keeping clotting in check
• Rat poison, blood clotting, and vitamin K
Protection against ingested foreign chemicals
• Cytochrome P450
• Secondary modification—addition of a polar group to
products of the P450 attack
• The response of the liver to the ingestion of foreignchemicals
• Multidrug resistance
Protection of the body against its own proteases
Protection against reactive oxygen species
• Mopping up oxygen free radicals with vitamins C and E
• Enzymic destruction of superoxide by superoxidedismutase
• The glutathione peroxidase-glutathione reductasestrategy
Further reading
Problems
Chapter 19 Nucleotide metabolism
Structure and nomenclature of nucleotides
• The sugar component of nucleotides
• The base component of nucleotides
Nomenclature
Structure of the bases
• Attachment of the bases in nucleotides
Synthesis of purine and pyrimidine nucleotides
• Purine nucleotides
PRPP-the ribotidation agent
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The one-carbon transfer reaction in purine nucleotide
synthesis
Where does the formyl group in formyl FH4 come from?
How are ATP and GTP produced from AMP and GMP?
• The purine salvage pathway
What is the physiological role of the purine salvage pathway?
• Formation of uric acid from purines
• Control of purine nucleotide synthesis
• Synthesis of pyrimidine nucleotides
• How are deoxyribonucleotides formed?
Conversion ofdUMP to dTMP
• Tetrahydrofolate, vitamin B12, and pernicious anemia
Further reading
Problems
Part 4 Information storageand utilization
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Chapter 20 DNA—its structure andarrangement in cells
What are nucleic acids?
The primary structure of DNA
• What are the bases in DNA?
• Attachment of the bases to deoxyribose
• What are the physical properties of the polynudeotidecomponents?
• Structure of the polynudeotide of DNA
• Why deoxyribose? Why not ribose?
• The DNA double helix
• DNA chains are antiparallel; what does this mean?
• How large are DNA molecules?
How is the DNA packed into a nucleus?
• How does the described structure of DNA correlate withthe compact eukaryotic chromosomes visible in the light
microscope?
• What is a gene in molecular terms?
• Some variations on the 'standard' gene
Repetitive DNA
• Where are we now?
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Further reading
Problems
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XX CONTENTS
Chapter 21 DNA synthesis and repair
Overall principle of DNA replication
Control of initiation of DNA replication in £ coli
Initiation and regulation of DNA replication in eukaryotes
Unwinding the DNA double helix and supercoiling
• How are positive supercoils removed ahead of thereplicative fork?
• What are the biological implications of topoisomerases?
• What is SSB?
The situation so far
The basic enzymic reaction catalysed byDNA polymerases
Problems in DNA synthesis
• How does a new strand get started?
• The polarity problem in DNA replication
• Mechanism of Okazaki fragment synthesis
• Enzyme complex at the replicative fork in E. coli
The DNA sliding clamp and the clamp loading mechanism
• What happens to the Okazaki fragments?
• Proofreading by polymerase III
• Methyl-directed mismatch repair
Repair of DNA damage in £ coli
• The machinery in the eukaryotic replicative fork
• The problem of replicating the ends of eukaryoticchromosomes
How is telomeric DNA synthesized?
DNA damage repair in eukaryotes
Is the mechanism described above the only way in
which DNA is synthesized?
Transposons or jumping genes
Homologous recombination
• Mechanism of homologous recombination in E. coli
Formation of cross-over junctions by single-strand invasionSeparation of the duplexes
• Recombination in eukaryotes
Further reading
Problems
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• The structure of RNA
• How is mRNAsynthesized?
• Some general properties of mRNA
• Some essential terminology
• A note on where we go from here
Gene transcription in E. coli
• What is specified by the term 'gene' in prokaryotes?• What do we mean by the 5' end of a gene?
• Phases of gene transcription
Initiation of transcription in E. coli
Untwisting the DNA
Termination of transcription
• The rate of gene transcription initiation in prokaryotes
• Control of transcription by different sigma factors
• How are individual E. coli genes regulated in a variablefashion? The lac operon
• Structure of the E. coli lac operon
• Control of transcription by attenuation
• The structure of DNA binding proteins
Helix-turn-helix proteins
Leucine zipper proteins
Zinc finger proteins
Further reading
Problems
Chapter 22 Gene transcription—the first stepin the mechanism by which genes directprotein synthesis
Messenger RNA
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Chapter 23 Eukaryotic gene transcription and
control
The basic processes involved in eukaryotic mRNAproduction
• Capping the RNA transcribed by RNA polymerase II
• What are split genes?
Mechanism of splicing
• What is the biological status of introns?
What is the origin of split genes?
Alternative splicing or two (or more) proteins for the price of onegene
Mechanism of initiation of eukaryotic gene transcriptionand its control
• Unpacking of the DNA for transcription
• A general overview of the differences in the initiation andcontrol of gene transcription in prokaryotes andeukaryotes
• Types of eukaryotic genes and their controlling regions
Type II eukaryotic gene promoters
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CONTENTS XXI
Enhancers and their mechanism of gene control: alternative models 362
Transcription factors 364
• How do activators promote transcriptional initiation? 364
The role ofchromatin in eukaryotic gene control 364
How do transcription factors open up gene promoters? 366
• How is transcription initiated on the opened promoter? 367
What is the mechanism by which transcription rates are controlledby the multiple transcription factors? 368
• How are activated genes switched off? 368
• Synthesis of the messenger RNA 369
• Termination of transcription in eukaryotes 369
mRNA stability and the control of gene expression 369
• Determinants of mRNA stability and their role in geneexpression control 370
Role of the poly A tail 370
Structural stability determinants ofmRNAs 370
Gene transcription in mitochondria 371
Genes that do not code for proteins 372
Ribozymes and self-splicing of RNA 372
• Self-cleaving in small circular RNA pathogens 373
• The hammerhead ribozyme mechanism of self-cleavage 374
• The concept of gene shears 374
Ribozymes and hypotheses about the origin of life 375
Further reading 376
Problems 378
Chapter 24 Protein synthesis 381
Essential basis of the process of protein synthesis 381
• The genetic code 381
• How are the codons translated? 382
• Transfer RNA 382
• The wobble mechanism 383
• How are amino acids attached to tRNA molecules? 384
Ribosomes 385
Initiation of translation 386
• Initiation of translation in E. coli 386
Once initiation is achieved, elongation is the next step 388
• Cytoplasmic elongation factors 388
• Mechanism of elongation 389
• How is fidelity of translation achieved? 390
Termination of protein synthesis 391
Physical structure of the ribosome 391
• What is a polysome? 392
How does protein synthesis differ in eukaryotes? 392
Protein synthesis in mitochondria 392
Effects of antibiotics and toxins on protein synthesis 393
How does the polypeptide chain synthesized on theribosome fold up? 394
• Chaperones (heat shock proteins) 394
Mechanism of action of molecular chaperones 394
• Enzymes involved in protein folding 395
Prion diseases and protein folding 396
A note on where we are in the book 397
Further reading 397
Problems 399
Chapter 25 How are newly synthesized proteinsdelivered to their correct destinations?—proteintargeting 403
A preliminary overview of the field 403
• Structure and function of the ER and Golgi apparatus 404
The important role of the GTP/GDP switch mechanism inprotein targeting 406
How are proteins secreted through the ER membrane? 406
• Mechanism of cotranslational transport through the ERmembrane 407
• Folding of the polypeptides inside the ER 408
• Glycosylation of proteins in the ER lumen and Golgiapparatus 408
How are proteins sorted, packaged, and despatched bythe Golgi apparatus? 409
Proteins to be returned to the ER 409
Proteins destined for lysosomes 409
Proteins to be secreted from the cell 409
• How are transport vesicles budded off from the Golgimembranes? 409
• How does the vesicle find the correct target membrane? 410
• How are membrane integral proteins inserted? 410
How is the membrane protein given the correct orientation? 411
Posttranslational transport of proteins into organelles 412
Transport of proteins into mitochondria 412
Targeting peroxisomal proteins 413
Nuclear-cytoplasmic traffic 414
Why should there be a nuclear membrane? 414
XXii CONTENTS
• The nuclear pore complex 414
• Nuclear localization signals 415
• Mechanism of nuclear-cytoplasmic transport and the roleof guanine nucleotide binding proteins 416
• Regulation of nuclear transport by cell signals and its rolein gene control 417
Further reading 418
Problems 420
Chapter 26 Cell signalling 423
What are the signalling molecules? 424
• Neurotransmitters 424
• Endocrine hormones 425
• Cytokines and growth factors 426
Growth factors and the celt cycle 426
• Vitamin D3 and retinoic acid 426
Intracellular receptor-mediated responses 427
Membrane receptor-mediated signalling systems 428
• An overview 428
Tyrosine kinase-associated receptors 430
• The Ras pathway 430
• Mechanism of the Ras signalling pathway 430
The Ras GTP/GDP switch mechanism 431
The protein kinase cascade of the Ras pathway 431
Nomenclature of the protein kinases of the Ras pathway 432
Inactivation and modulation of the Ras pathway 432
Multiplicity of MAP kinase pathways 433
• The phosphatidylinositide 3 (PI3)-kinase pathway andinsulin signalling 433
• The JAK/STAT pathways; direct intracellular signallingpathways from receptor to nucleus 434
Vision: a process dependent on a G-protein-coupled
The G-protein-coupled receptors
• Overview
• Structure of G protein receptors
• Epinephrine signalling—a G protein pathway mediated bycAMP as second messenger
Control ofcAMP levels in cells
Different types of epinephrine receptors
How does cAMP control gene activities?
Desensitization of the G protein receptors
* G-protein-coupled receptors which work via a differentsecond messenger; the phosphatidylinositol cascade
• Other control roles of calcium
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receptor
Transduction of the light signal
Signal transduction pathways using cGMP as secondmessenger
• Activation of a guanylate cyclase by nitric oxide
Further reading
Problems
Chapter 27 The immune system
• The cells involved in the immune system
• There are two immune protective systems
• Where is the immune system located in the body?
Antibody-based or humoral immunity
• Structure of antibodies
• What are the functions of antibodies?
• What are the different classes of antibodies?
• Generation of antibody diversity
Activation of B cells to produce antibodies
• Deletion of potentially self-reacting B cells in the bonemarrow
• The theory of clonal selection
• What is the mechanism by which B cells are activated tosecrete antibody?
Role of helper T cells
Activation of helper T cells
Activation ofB cells by activated helper T cells
• Affinity maturation of antibodies
• Memory cells
T cells and cell-mediated immunity
• Activation of cytotoxic T cells
• Mechanism of action of cytotoxic cells
Why does the human immune system so fiercely rejectforeign human cells?
Monoclonal antibodies
Further reading
Problems
Chapter 28 Viruses and viroids
The life cycle of a virus
• Types of genetic material in different viruses
• How are viruses released from cells?
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Replication mechanisms of some selected viruses
• Vaccinia
• Poliovirus
• Influenza virus
•. Retroviruses
• Cancer-inducing, or oncogenic, retroviruses
• Bacteriophage lambda
What are viroids?
Further reading
Problems
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Chapter 29 Gene cloning, recombinant DNAtechnology, genetic engineering 477
• What were the problems in isolating genes? 477
The first step-cutt ing the DNA with restriction
endonucleases 477
• What is the biological function of restriction enzymes? 478
Gene cloning, or how genes are isolated 478
• What does 'cloning' mean? 478
Isolation of a genomic clone of a human gene 479
• Preparation of a human gene library 479
• How are recombinant molecules constructed? 479
• Screening of the plaques for the desired human gene 481
Cloning a human cDNA 481
• Preparation of a human cDNA library 481
What can be done with the cloned DNA? 481
• Bacterial plasmids 482
• Determination of the base sequence of a cloned piece ofDNA 483
Outline of the dideoxy DNA-sequencing technique 483
How is all this interpreted as a base sequence? 483
• DNA databases and genomics 485
• The polymerase chain reaction (PCR) for amplifying aspecific DNA segment 485
• Applications of recombinant DNA technology 486
Expression of the gene in bacterial and eukaryotic hosts 486
Site-directed mutagenesis 486
• Transgenesis and gene therapy 487
Knockout mice or gene targeting 487
• The embryonic stem (ES) cell system 488
• Gene targeting 488
• Detection of genetic abnormalities by restriction analysisor Southern blotting 491
CONTENTS
Further reading
Problems
Chapter 30 Molecular biology of cancer
The cell cycle and its control
• Overview of the cycle
• Control of the cell cycle
How do growth factors and cytokines cause the transition past the
restriction checkpoint in G,?
What is the molecular nature of cancer?
• What types of genetic changes are involved in generationof the cancerous state?
How are oncogenes acquired?
How do oncogenes cause cancer?
• The central role of signal transduction
What are the roles of tumour suppressor genes?
Further reading
Problems
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Part 5 Transport of oxygenand C02
Chapter 31 The red blood cell and the role ofhemoglobin 507
The red blood cell 507
Heme and its synthesis 508
• Synthesis of heme 508
• Regulation of heme biosynthesis and iron supply to theerythrocyte 509
• Destruction of heme 511
How is globin synthesis regulated? 511
Transport of gases in the blood 513
• What is the chemical rationale for a heme-protein
complex as the oxygen carrier? 513
• Structure of myoglobin and its oxygen binding role 513
• Structure of hemoglobin 513
• Binding of oxygen to hemoglobin 513
• How is the sigmoidal oxygen saturation curve achieved? 513
• Mechanism of the allosteric change in hemoglobin 514
• The essential role of 2:3-bisphosphoglycerate (BPG) inhemoglobin function 516
XXiv CONTENTS
• Effect of pH on oxygen binding to hemoglobin
• Role of pH changes in oxygen and CO2 transport
• pH buffering in the blood
Sickle cell anemia
Further reading
Problems
Part 6 Mechanical workby cells
Chapter 32 Muscle contraction
• A reminder of conformational changes in proteins
Types of muscle cells and their energy supply
• Structure of skeletal striated muscle
Structure of the myo fibril
How does the sarcomere shorten?
Structure and action of thick and thin filaments
• How does the myosin head convert the energy of ATPhydrolysis into mechanical force on the actin filament?
Mechanism of the conformational changes in the myosin head
How is contraction in voluntary striated musclecontrolled?
• How does Ca2+ trigger contraction?
Release and uptake of Co** in the muscle
How does smooth muscle differ in structure and controlfrom striated muscle?
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• Control of smooth muscle contractions 532
How does Ca2* control smooth muscle contraction? 532
Further reading 532
Problems 533
Chapter 33 The cytoskeleton, molecular
motors, and intracellular transport 535
An overview 535
The role of actin and myosin in nonmuscle cells 536
• Structural role of actin and its involvement in cell
movement 536
• The role of actin and myosin in intracellular transport ofvesicles 537
Microtubules, cell movement, and intracellular transport 538
• What are microtubules? 538
What protects the positive (+) ends of microtubules? 538
• Functions of microtubules 539
Molecular motors: kinesins and dyneins 539
Role of microtubules in cell movement 539
Role of microtubules in vesicle transport inside the cell 540
Role of microtubules in mitosis 540
Intermediate filaments 540
Further reading 541
Problems 542
Answers to problems 555
Figure acknowledgements 571
Index 573