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

21

21

22

23

24

24

26

26

26

26

26

27

27

27

27

27

7

8

8

8

11

11

12

12

13

14

15

15

17

18

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

28

29

33

33

35

35

36

36

36

37

3738

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

109

109

109

109

109

110

110

111

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

135

136

137

137

137

138

138

139

139

139

140

140

142

142

143

145

147

147

147

147

147

149

150

150

151

153

153

154

154

154

155

156

156

• 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

181

182

182

183

183

184

184

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

187

187

187

188

188

188

189

189

190

191

192

193

194

195

195

196

196

197

197

201

201

202

203

203

• 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

226 Further reading

Problems227

249

250

228

228

228

229

229

230

231

231

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

239

239

241

241

241

242

243

244

244

245

246

246

246

247

247

248

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

267

267

268

269

270

270

271

271

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

277

277

277

278

279

279

280

280

281

281

281

282

282

283

283

283

284

285

287

287

287

288

288

288

288

289

289

289

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

290

293

294

294

294

295

295

296

296

297

298

299

299

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?

303

303

303

304

304

304

304

305

306

308

310

310

312

312

313

313

314

Further reading

Problems

314

314

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

317

317

318

318

319

320

321

322

323

323

324

324

324

325

325

326

327

328

329

330

333

333

334

335

335

335

336

336

338338

339

340

341

• 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

343

343

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

343

343

344

345

345

346

346

346

346

346

347

347

348

348

348

349

351

351

352

354

354

354

355

357

357

357

357

358

359

359

360

360

360

361

362

362

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

436

436

436

436

437

438

438

439

439

440

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?

441

442

443

444

445

448

451

451

452

452

452

452

453

453

453

455

455

455

455

456

456

456

457

457

458

458

459

460

461

461

462

465

465

466

467

Replication mechanisms of some selected viruses

• Vaccinia

• Poliovirus

• Influenza virus

•. Retroviruses

• Cancer-inducing, or oncogenic, retroviruses

• Bacteriophage lambda

What are viroids?

Further reading

Problems

468

468

468

469

470

471

471

472

473

473

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

xxiii

491

492

495

496

496

497

498

498

498

499

500

500

501

502

503

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?

517

517

518

518

518

519

523

523

523

524

524

524

525

526

526

530

530

530

531

• 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