Multiple Aspects of DNA and RNA: from Biophysics to Bioinformatics (eBook)

Previous sessions 7
Lecturers 10
Short lectures and seminar speakers 11
Organizers 11
Participants 12
Preface 16
Contents 18
DNA structure, dynamics and recognition 26
Introduction to the DNA double helix 30
Biophysical studies of DNA - structure and stability 41
DNA dynamics 46
Deformations of the double helix 50
DNA recognition 58
Introduction to non-Watson–Crick base pairs and RNA folding 66
Definitions 70
The annotation of non-Watson-Crick base pairs and of RNA motifs 77
RNA-RNA recognition motifs 81
Roles of RNA motifs in RNA-protein recognition 86
Conclusions 94
References 94
Regulation of transcription by RNA polymerase II 98
Introduction 102
DNA regulatory elements 104
Basal/general transcription factors 106
Sequence-specific DNA-binding factors 108
Chromatin and transcription 110
Conclusions and speculations 112
References 114
Basic concepts of statistical physics of polymers 116
Introduction to polymer physics 120
Fundamentals of physical viewpoint in polymer science 120
Flexibility of a polymer chain. Flexibility mechanisms 121
Rotational-isomeric flexibility mechanism 121
Persistent flexibility mechanism 123
Freely-jointed flexibility mechanism 123
Types of polymer molecules 123
Physical states of polymer materials 125
Polymer solutions 126
Single ideal polymer chain 127
Definition of ideal polymer chain 127
Size of ideal freely-jointed chain. Entangled coil 127
Size of ideal chain with fixed valency angle 129
Kuhn segment length of a polymer chain 130
Persistent length of a polymer chain 131
Stiff and flexible chains 132
Gaussian distribution for the end-to-end vector for ideal chain 133
High elasticity of polymer networks 134
The property of high elasticity 134
Elasticity of a single ideal chain 136
Elasticity of a polymer network (rubber) 138
Viscoelasticity of entangled polymer fluids 141
Main properties of entangled polymer fluids 141
Viscosity of fluids 142
The property of viscoelasticity 143
Theory of reptations 144
The method of gel-electrophoresis in application to DNA molecules 147
Gel permeation chromatography 149
References 150
The physics of DNA electrophoresis 152
Importance of DNA sorting in biology and how physics can help 156
Physical description of DNA 158
Electrophoretic force 159
DNA sequencing: gel electrophoresis of single-stranded DNA 160
Reptative dynamics 161
Biased reptation 162
Repton model 165
Strategies for DNA sequencing 166
Gel electrophoresis of long double-stranded DNA molecules 167
Complex dynamics in constant fields 167
Pulsed-field gel electrophoresis: separation of restriction fragments 169
Difficulty of separating very large molecules 172
Obstacle courses on microchips 173
Collision of a DNA molecule with an obstacle 174
Efficient pulsed-field fractionation in silicon arrays 175
Continuous separation in asymmetric pulsed fields 177
Asymmetric sieves for sorting DNA 179
Rapid continuous separation in a divided laminar flow 181
Summary 183
References 183
Single-molecule studies of DNA mechanics and DNA/protein interactions 186
Introduction 190
The interest of physicists for DNA 191
Ease of handling 191
DNA as a model polymer 192
Introduction to single-molecule DNA manipulation techniques 193
Strategies and forces involved 193
Measurement techniques 195
Force measurements 197
Measuring forces with Brownian motion 197
Advantages and disadvantages of the manipulation techniques 199
Mechanical properties and behavior of DNA 200
Tertiary structures in DNA 200
Topological formalism 201
DNA supercoiling in vivo 202
DNA unwinding and helix destabilisation 202
DNA topoisomerases 202
Supercoiling and transcription 203
DNA elasticity in the absence of torsion (sigma=0) 203
Results 204
Theoretical models 205
Mechanical properties of supercoiled DNA 205
Results 206
Interpretation 206
The buckling instability in DNA 208
Stretching single-strand DNA 209
Conclusions on the mechanical properties of nucleic acids 210
RNA polymerases 211
An introduction to transcription 211
Historical overview: transcription elongation, or RNA polymerase as a linear motor 213
RNA polymerase as a torquing device: the case of transcription initiation 214
DNA topoisomerases 218
Type I and Type II topoisomerases 218
Eukaryotic topoisomerase II 220
Enzymatic cycle 220
Prokaryotic topoisomerase IV 221
Experimental results: D. melanogaster topoisomerase II 221
Calibrating the experiment 221
Crossover clamping in the absence of ATP 222
Low ATP concentrations: detecting a single enzymatic cycle 223
High ATP concentration 224
Determining Vsat, the saturated reaction velocity 225
Effect of the stretching force 226
Relaxation of negatively supercoiled DNA 227
Topo II only removes crossovers in DNA 227
A comparison with E. coli topo IV 228
Experiments on braided DNA molecules 229
Conclusions on type II topoisomerases 229
Conclusions and future prospects 230
References 230
Introduction to single-DNA micromechanics 236
Introduction 240
The double helix is a semiflexible polymer 242
Structure 242
DNA bending 244
Discrete-segment model of a semiflexible polymer 244
Bending elasticity and the persistence length 246
End-to-end distance 247
DNA loop bending energies 248
Site-juxtaposition probabilities 249
Permanent sequence-driven bends 250
Stretching out the double helix 250
Small forces (< kB T/A = 0.08 pN)
Larger forces (> kB T/A = 0.08 pN)
Free energy of the semiflexible polymer 254
Really large forces (> 10 pN)
Strand separation 256
Free-energy models of strand separation 257
Sequence-dependent models 258
Free energy of internal `bubbles' 259
Small internal bubbles may facilitate sharp bending 260
Stretching single-stranded nucleic acids 261
Unzipping the double helix 263
Effect of torque on dsDNA end 265
Fixed extension versus fixed force for unzipping 266
DNA topology 267
DNA supercoiling 267
Twist rigidity of the double helix 267
Writhing of the double helix 268
Simple model of plectonemic supercoiling 269
Twisted DNA under tension 273
Forces and torques can drive large structural reorganizations of the double helix 275
DNA knotting 276
Cells contain active machinery for removal of knots and other entanglements of DNA 277
Knotting a molecule is surprisingly unlikely 277
Condensation-resolution mechanism for disentangling long molecules 278
DNA-protein interactions 279
How do sequence-specific DNA-binding proteins find their targets? 280
Three-dimensional diffusion to the target 280
Nonspecific interactions can accelerate targeting 281
Single-molecule study of DNA-binding proteins 282
DNA-looping protein: equilibrium `length-loss' model 282
Loop formation kinetics 283
DNA-bending proteins 284
Analytical calculation for compaction by DNA-bending proteins 287
Effects of twisting of DNA by proteins 289
Surprising results of experiments 290
Conclusion 291
References 293
The analysis of regulatory sequences 296
Forewords 300
Scope of the course 300
Web site and practical sessions 300
Transcriptional regulation 300
The non-coding genome 300
Transcriptional regulation 301
Representations of regulatory elements 304
String-based representations 304
Matrix-based representation 305
Pattern discovery 308
Introduction 308
Study cases 308
String-based pattern discovery 309
Analysis of word occurrences 309
Analysis of dyad occurrences (spaced pairs of words) 316
Strengths and weaknesses of word- and dyad-based pattern discovery 318
String-based pattern matching 319
Matrix-based pattern discovery 321
Consensus: a greedy approach 322
Gibbs sampling 322
Strengths and weaknesses of matrix-based pattern discovery 324
Concluding remarks 326
Practical sessions 327
Appendices 327
IUPAC ambiguous nucleotide code 327
A survey of gene circuit approach applied to modelling of segment determination in fruitfly 330
Preamble 334
Introduction 334
The biology of segment determination 335
Method description 336
The gene circuit modelling framework 336
Quantitative expression data 338
Optimization by Parallel Lam Simulated Annealing (PLSA) and Optimal Steepest Descent Algorithm (OSDA) 339
Selection of gene circuits 339
Software and bioinformatics 340
Analysis of regulatory mechanisms controlling segment determination 340
Regulatory interactions in gap gene system 340
Stripe forming architecture of the gap gene system 343
Pattern formation and nuclear divisions are uncoupled in Drosophila segmentation 343
Conclusions 345
References 346
Modeling, analysis, and simulation of genetic regulatory networks: from differential equations to logical models 350
Introduction 354
Ordinary differential equations 356
Models and analysis 356
Analysis of regulatory networks involved in cell-cycle control, circadian rhythms, and development 359
Piecewise-linear differential equations 360
Models and analysis 360
Simulation of the initiation of sporulation in Bacillus subtilis 364
Logical models 368
Models and analysis 368
Modeling of the lysis-lysogeny decision during the infection of Escherichia coli by bacteriophage lambda 370
Extensions of logical modeling 374
Conclusions 375
References 376