Methods in Enzymology, Vol. 371: RNA Polymerases and Associated Factors, Part D

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Model for Polymerase Translocation......Page 31
Kinetic Consequences and Tests of the Model......Page 33
Using Exonucleases to Study Translocation......Page 36
Future Studies......Page 39
Acknowledgments......Page 41
Evaluation of Fluorescence Spectroscopy Methods for Mapping Melted Regions of DNA Along the Transcription Pathway......Page 42
Benefits of Fluorescence over Other Approaches......Page 44
Preparation of Samples......Page 46
Considerations......Page 48
Fluorescent Probes Report Only Indirectly on Melting......Page 49
Mapping the Bubble in the Initial Promoter Complex......Page 51
Mapping the Bubble in a Stably Stalled Elongation Complex......Page 53
Monitoring Bubble Collapse on Promoter Clearance......Page 56
Monitoring Initial Dissociation of the 5' End of the RNA......Page 58
Concluding Remarks......Page 60
Acknowledgments......Page 62
Single DNA Molecule Analysis of Transcription Complexes......Page 63
Surface Equilibration of DNA Molecules......Page 64
Sample Preparation......Page 66
DNA Contour Length Measurements......Page 67
DNA Bend-Angle Determination......Page 70
DNA Wrapping in Transcription Complexes......Page 73
Tagging a specific RNA polymerase subunit......Page 76
Concluding Remarks......Page 78
Acknowledgments......Page 79
Assay for Movements of RNA Polymerase along DNA......Page 80
Assay for Sliding by Single-Molecule Dynamics......Page 82
Multiple Introduction of Tetramethylrhodamine Into Avidin......Page 83
Preparation of Fluorescently Labeled RNA Polymerase......Page 85
Fixing of Extended DNA in Parallel......Page 86
Preparation of a Slide with Electrodes......Page 88
Assay and Analysis of Sliding Motion......Page 89
Detection of Rotational Motion and its Application for Groove Tracking During Sliding23......Page 90
Preparation of a Rotational Indicator......Page 91
Assay of Groove Tracking During Sliding......Page 93
Kinetic Assay for Moribund Complex8-10,27......Page 94
Isolation and Examination of Moribund and Dead-End Complex9,10,27......Page 96
Single-Molecule Based Techniques Used in Elongation......Page 97
Cautions of Application of Single-Molecule Techniques......Page 99
Formation of Stalled Elongation Complexes......Page 101
Rapid Quench Kinetic Experiments......Page 103
Quantification and Normalization of Rate Data......Page 104
Single and Double Exponential Fits......Page 105
Determination of the Final Kinetic Mechanism......Page 109
Principles and Methods of Affinity Cleavage in Studying Transcription......Page 112
Obtaining Bifunctional Chelating Agents......Page 113
Principles and Practical Aspects......Page 114
Conjugation of FeBABE to a Single Site......Page 116
Conjugation to Multiple Sites......Page 117
Conjugation to DNA......Page 120
Conjugation Yield......Page 121
Cleavage of RNAP Holoenzyme by lacUV5-FeBABE Conjugate......Page 123
Preparing the Protein......Page 125
Quenching......Page 127
Markers......Page 128
Peptide-Terminus Specific Antibodies......Page 129
Comparison with Structural Data......Page 130
Sigma/Core Interactions Using Single-Cys Mutants of Sigma......Page 131
Sigma/DNA Interactions Using Single-Cys Mutants of Sigma......Page 132
DNA/Holoenzyme Interactions Using Proteolytic DNA......Page 134
Iodine-⁄⁄125 Radioprobing of E. coli RNA Polymerase Transcription Elongation Complexes......Page 137
Experimental Scheme......Page 138
Synthesis of [125I]ICTP......Page 144
Assembly of Transcription Elongation Complex and RNAP "Walking"......Page 145
Analysis of Breaks in DNA......Page 146
Breaks in the T-strand and N-strand of DNA......Page 147
Distribution of Breaks in the T-strand......Page 148
DNA Conformation in RNAP Active Center......Page 149
Concluding Remarks......Page 150
Acknowledgments......Page 151
Formation of Long DNA Templates Containing Site-Specific Alkane-Disulfide DNA Interstrand Cross-Links for Use in Transc......Page 152
Convertible Nucleosides......Page 153
Construction of Templates with Site-Specific Interstrand DNA Cross-Links......Page 155
Preparation of Disulfide Cross-linked Templates......Page 157
Purification of Pure Cross-linked Template......Page 159
Transcription Conditions for Fig. 4A......Page 160
Results......Page 161
Acknowledgment......Page 164
Probing the Organization of Transcription Complexes Using Photoreactive 4-Thio-Substituted Analogs of Uracil and Thymidine......Page 165
RNA Polymerase, Nucleotide Analogs, and Template Assembly......Page 166
Preparation of Transcription Complexes with Specifically Positioned Photoreactive Analogs......Page 167
Template Design and Construction......Page 168
Activation of the Cross-Link and Analysis of the Sample......Page 171
Resolution and Purification of Cross-Link Products......Page 172
Cleavage and Mapping......Page 173
Acknowledgments......Page 175
Fluorescence Resonance Energy Transfer (FRET) in Analysis of Transcription-Complex Structure and Function......Page 176
Preparation of DNA Fragments......Page 177
Preparation of s70......Page 179
Preparation of RNAP Core......Page 181
Preparation of RNA Polymerase-Promoter Open Complex (RPo)......Page 184
DNA-RNAP FRET (Trailing-Edge/Leading-Edge FRET)......Page 185
Core-s70 FRET......Page 187
DNA-RNAP FRET (Trailing-Edge/Leading-Edge FRET)......Page 188
Core-s70 FRET......Page 190
Acknowledgments......Page 191
Methods of Walking with the RNA Polymerase......Page 192
General Considerations......Page 193
Preparation of RNAP......Page 194
Preparation of NTP Substrates......Page 196
Preparation of the DNA Template......Page 197
His-Tagged-Based Walking......Page 198
Biotin-Tagged-Based Walking......Page 199
Roadblocking......Page 200
Acknowledgment......Page 201
Stepwise Walking and Cross-Linking of RNA with Elongating T7 RNA Polymerase......Page 202
Biotinylated DNA Template......Page 203
Purification of T7 RNA Polymerase......Page 204
Stepwise Walking of the T7 Transcription Complex......Page 205
Mapping the RNA Cross-Links in Elongating RNA Polymerase......Page 208
Acknowledgments......Page 211
Characterization of Protein-Nucleic Acid Interactions that are Required for Transcription Processivity......Page 212
Determination of the Minimal DNA Template Needed for EC Stability......Page 213
Template Switching......Page 214
Analysis of EC Stability at the End of the DNA Template......Page 218
Determination of the Minimal RNA Product Needed for EC Stability......Page 219
Generation of EC6-EC11 and Assessing Their Stability......Page 220
Determination of the Minimal Size of the RNA:DNA Heteroduplex Needed for EC Stability......Page 221
Acknowledgments......Page 223
Strategies and Methods of Cross-Linking of RNA Polymerase Active Center......Page 224
Labeling of the Priming Substrate Binding Site of the Active Center by Autocatalysis......Page 225
RNA-Protein Cross-linking in the Active Center of Initiating Complex......Page 226
Cross-linking Protocol for TEC219......Page 228
Mapping of Cross-linking Sites......Page 229
CNBr Degradation at Met Residues17......Page 232
Detection and Mapping of Multiple Cross-linking Sites......Page 233
Refining the Mapping......Page 236
Acknowledgment......Page 239
Using a lac Repressor Roadblock to Analyze the E. Coli Transcription Elongation Complex......Page 240
Effect of a lac Repressor Roadblock on Expression of a Downstream Gene in vivo......Page 244
Effect of a lac Repressor Roadblock on Transcription in vitro......Page 245
Protocol for Roadblocking the TEC with lac Repressor......Page 248
Concluding Remarks......Page 251
Biochemical Assays of Gre Factors of Thermus Thermophilus......Page 252
Procedure 1......Page 255
Procedure 2......Page 258
Assays for Inhibitory Activity of GreA2......Page 260
Procedure 3......Page 261
Localized Fe2+-Induced Hydroxyl Radical Mapping of GreA1 and GreA2 Sites Proximal to RNAP Catalytic Center......Page 263
Acknowledgments......Page 265
Engineering of Elongation Complexes of Bacterial and Yeast RNA Polymerases......Page 266
Buffers, Enzymes, and Reagents......Page 267
Promoter DNA Templates and RNA/DNA Oligonucleotides......Page 268
Obtaining Defined ECs from Promoters: Basic Protocol......Page 269
Comments......Page 271
Truncation of Nascent RNA from 5' End in the EC with RNase T1 and RNase A......Page 272
Comments......Page 274
Isolation of the EC with 8-10-nt RNA by Treatment with Pyrophosphate and GreB......Page 275
Assembly of Functional EC of E. coli RNAP and S. cerevisiae Pol II from Synthetic DNA and RNA Oligonucleotides......Page 276
Assembly of EC with Histidine-Tagged Bacterial RNAP and Yeast Pol II and Immobilization of the EC on Ni2+-NTA Agarose Be......Page 278
Comments......Page 281
Basic Protocol......Page 282
Assembly and Purification of Large Quantities of the E. coli EC in Solution......Page 283
Assay of Transient State Kinetics of RNA Polymerase II Elongation......Page 285
Preparation of Precisely Stalled Elongation Complexes on Bead Templates......Page 286
Rapid Quench Experiments......Page 288
Elongation from C40......Page 290
The Running-Start, Two-Bond Assay......Page 294
NTP-Dependent Steps in Transcription......Page 295
Conclusion......Page 296
Acknowledgments......Page 297
Analysis of RNA Polymerase II Elongation In Vitro......Page 298
Preparation of Nuclear Extract......Page 300
Pulse-Chase Assay......Page 302
Preparation of Immobilized Templates......Page 304
Isolation of Elongation Complexes......Page 305
Add-Back Assay......Page 307
Acknowledgment......Page 309
Introduction......Page 310
Preparations of ELL......Page 312
Preparation of the Recombinant Elongin ABC Complex in E. coli......Page 313
Assays of Elongin and ELL......Page 316
Acknowledgments......Page 317
History and Rationale......Page 318
Mycophenolic acid......Page 320
Induction of IMP Dehydrogenase......Page 321
Conventional Chromatography (after Edwards et al., 1990)2......Page 322
Affinity Chromatography......Page 323
Acknowledgments......Page 326
Acetylation of Human AP-Endonuclease 1, A Critical Enzyme in DNA Repair and Transcription Regulation......Page 327
Assay Procedure......Page 328
General Considerations......Page 329
Identification of Acetylacceptor Lys Residues in AcAPE1......Page 331
Electrophoretic Mobility Shift Assay with nCaRE-B......Page 332
Repression of Reporter Gene Expression Dependent on APE1 Acetylation......Page 333
Acknowledgments......Page 335
Characterization of Transcription-Repair Coupling Factors in E. Coli and Humans......Page 336
Protein Purification......Page 338
Substrates......Page 340
DNA Constructs for Expression......Page 341
Protein Purification......Page 343
Transcription-Repair Coupling Factor (TRCF) and Mutation Frequency Decline (Mfd)......Page 344
Primary Structure of the TRCF......Page 345
Transcription-Stimulated Repair by TRCF: Repair Synthesis Assay......Page 346
TRCF Binding to UvrA and RNAP......Page 349
Removal of Stalled RNAP by TRCF......Page 351
Removal of Arrested RNAP by TRCF......Page 352
Structure and Function of CSB......Page 353
Failure of CSB to Dissociate Stalled RNAPII......Page 354
Effect of CSB on Elongating and Stalled RNAPII......Page 355
"Passive" Model for Human Transcription-Coupled Repair......Page 357
Dissociation of Stalled RNAPII by Transcription Factor 2......Page 358
Comparison of TRCF and CSB......Page 359
Acknowledgments......Page 360
Techniques to Analyze the HIV-1 Tat and TAR RNA-Dependent Recruitment and Activation of the Cyclin T1:CDK9 (P-TEFb) Transcrip......Page 361
Preparation of Recombinant HIV-1 Tat Protein......Page 366
In Vitro Transcription Assay with HIV-1 Tat......Page 367
HIV-1 TAR RNA Affinity Chromatography......Page 369
RNA Gel Mobility Shift Analysis of Tat:CycT1:CDK9 Complexes......Page 371
Protocol for Purification of Native Cyclin T1 from Nuclear Extracts by GST-Tat Pull-Down Affinity Selection......Page 372
Acknowledgments......Page 373
Assay of Intrinsic Transcript Termination by E. Coli RNA Polymerase on Single-Stranded and Double-Stranded DNA Templates......Page 374
Buffers......Page 375
His6-Tagged RNA Polymerase Purification......Page 376
Solid Phase Transcription and Transcript Termination in Vitro......Page 378
Buffers......Page 381
Protocol......Page 382
Termination Assay......Page 383
Use of Chemiluminescence or Fluorescence rather than Radioisotopes for Detection of ExoIII Digestion......Page 384
Acknowledgments......Page 386
Bacteriophage HK022 Nun Protein: A Specific Transcription Termination Factor that Excludes Bacteriophage l......Page 387
Induction......Page 388
Purification......Page 389
Assay of Nun Protein......Page 391
Assay of Transcription Termination by Ribosomal Protein L4......Page 392
Construction of Reporter Genes......Page 393
Basic [35S]Methionine Labeling Protocol......Page 395
Preparing and Running an SDS-Polyacrylamide Gel......Page 397
In Vitro Assay of L4-Mediated Transcription Termination......Page 398
Purification of DNA Templates......Page 399
Design of the Transcription Reaction......Page 400
Recipes for in Vitro Transcription......Page 401
Single-Round Transcription Reaction (40 ml Final Volume)......Page 402
Purification of Paused Transcription Complexes and Restart of Transcription......Page 403
Transcript Release Filtration Assay......Page 404
DNA Competition Experiments......Page 405
Analysis of the Intrinsic Transcription Termination Mechanism and Its Control......Page 406
The Minimal Termination and Antitermination System......Page 407
Obtaining and Analyzing the Trapped Intermediate in Termination......Page 411
Pausing at the Termination Point: Detection and Functional Analysis......Page 415
The "Slow Termination" Assay......Page 418
Acknowledgments......Page 419
Introduction......Page 420
Preparation of Active S-30 Extracts, and Standard Reaction Conditions and Procedures......Page 422
Construction of Circular DNA Templates Suitable for in Vitro Analysis......Page 423
Measurement of in Vitro Rho-Dependent Termination and its Prevention......Page 424
Measuring Ribosome Stalling at the tnaC Stop Codon and Peptidyl-tRNA Accumulation......Page 426
Identification of the tRNA of the Peptidyl-tRNA in the Stalled Ribosome......Page 427
Comments and Variations......Page 428
Role of RNA Structure in Transcription Attenuation in Bacillus subtilis: The trpEDCFBA Operon as a Model System......Page 429
Prediction of RNA Secondary Structure......Page 430
Enzymatic and Chemical Probes......Page 431
Enzymatic and Chemical Probing......Page 432
In vivo Demonstration That RNA Structure Participatesin Attenuation......Page 435
In Vitro Demonstration That RNA Structure Participates in Attenuation......Page 436
Multi-round in vitro Transcription Assay......Page 437
Single-round in vitro Transcription Assay......Page 438
Background......Page 442
Shiga Toxin......Page 443
Analysis: N-mediated Antitermination......Page 445
Using the DNA Sequence to Identify Components of the N Antitermination System......Page 446
Use of E. coli and H-19B Mutants to Analyze N-mediated Antitermination......Page 448
Use of Reporter Constructs to Analyze N-mediated Antitermination......Page 451
Use of Reporter Constructs to Analyze the nut Region......Page 452
Use of Reporter Constructs to Analyze Q-mediated Antitermination......Page 455
Q Action and stx Gene Expression......Page 457
Phage Induction and Stx Release......Page 458
Analysis of Promoters Directly Involved in Stx Production......Page 459
Some Considerations......Page 461
Acknowledgments......Page 462
Introduction......Page 463
Transcription Antitermination in Lambdoid Phages......Page 464
Experimental Strategies......Page 467
Experimental Procedures......Page 473
Acknowledgment......Page 484
Assay of Transcription Antitermination by Proteins of the CspA Family......Page 485
Expression and Purification of CspE......Page 487
Transcription Antitermination In Vivo......Page 488
Transcription Antitermination In Vitro......Page 490
Properties of CspA Homologs Important for Transcription Antitermination......Page 491
Comments......Page 496
Assay of Antitermination of Ribosomal RNA Transcription......Page 497
NusA......Page 498
NusB......Page 499
NusE......Page 501
NusG......Page 502
Ribosomal Protein S4......Page 503
Purification of EcoRI*......Page 504
Preparation of AD7333 S100 Extracts......Page 505
In Vitro Transcription Reactions......Page 506
Isolation of Antitermination Complexes......Page 510
Introduction......Page 513
Procedure......Page 514
Materials and Reagents......Page 516
Procedure......Page 517
Purification of Elongating RNA Polymerase II......Page 518
Procedure......Page 519
Chromatin Assembly In Vitro with Purified Recombinant ACF and NAP-1......Page 521
Reagents and Equipment......Page 522
Drosophila NAP-1......Page 523
Drosophila ACF......Page 524
Drosophila Topoisomerase I Catalytic Domain (ND423)......Page 526
Reagents and Equipment......Page 527
ATP Regeneration System and Relaxed DNA......Page 528
Reagents and Equipment......Page 529
Micrococcal Nuclease Digestion Assay......Page 530
Optimization of the Assembly Reaction......Page 532
DNA Supercoiling Assay......Page 533
Assembly of Chromatin with the Linker Histone H1......Page 534
Micrococcal Nuclease Digestion Analysis of H1-containing Chromatin......Page 535
Acknowledgments......Page 537
Preparation of tTGase from human erythrocytes......Page 538
TTGase Activity Assay......Page 539
In vitro cross-linking reaction......Page 540
Cross-Linked Histone Subunits......Page 541
Perpectives......Page 542
Chromatin Decompaction Method by HMGN Proteins......Page 544
Preparation of Nucleosome Core Particles and Other Chromatin Substrates......Page 545
Gel Shift Assay......Page 546
Additional Approaches to Study the Interaction of HMGN Proteins with Nucleosomes......Page 547
Demonstrating an Interaction of HMGN with Minichromosomes......Page 548
Unfolding of Minichromosomes......Page 549
Association of HMGN Proteins with Chromatin In Vivo......Page 551
Chromatin Unfolding by HMGN Proteins In Vivo......Page 552
Nuclear Isolation......Page 553
Micrococcal Nuclease Digestion of Isolated Nuclei......Page 554
Analysis......Page 555
Interaction with Core Histone Tails......Page 557
Counteracting Linker Histone Compaction......Page 558
Concluding Remarks......Page 559
Assay of Activator Recruitment of Chromatin-Modifying Complexes......Page 560
Material and Reagents......Page 561
Procedure......Page 562
Procedure......Page 564
Immobilization of Nucleosomal G5E4-5S Template on Paramagnetic Beads......Page 565
In Vitro Recruitment Assay......Page 566
Procedure......Page 567
Insights into Structure and Function of GCN5/PCAF and yEsa 1 Histone Acetyltransferase Domains:......Page 569
The GCN5/PCAF HAT family......Page 570
The MYST HAT Family and Yeast Esa1......Page 571
Structure of the GCN5/PCAF HAT domain......Page 573
Structure of yEsa1......Page 574
X-ray Crystallographic Studies to Probe the Catalytic Mechanism of GCN5/PCAF......Page 576
Enzymatic Assays of GCN5/PCAF HAT Domains Support an Ordered-Sequential Reaction Mechanism and the Role of yGcn5 Glu-......Page 577
Bi-substrate Analog Inhibitor Studies with the GCN5/PCAF HAT Domain......Page 581
Substrate Specificity of GCN5/PCAF......Page 582
X-ray Crystallographic Studies to Probe the Catalytic Mechanism of yEsa1......Page 583
Biochemical Assays Reveal a Ping-Pong Catalytic Mechanism for Esa1......Page 584
Conclusions......Page 587
Assay of the Fate of the Nucleosome During Transcription by RNA Polymerase II......Page 589
Template Preparation......Page 590
Reconstitution, characterization, and purification of mononucleosomes and hexasomes......Page 591
Nucleosomes and hexasomes reconstituted from purified histones......Page 593
Transcription Buffers......Page 594
EC assembly and ligation to DNA or nucleosomal templates......Page 595
Transcript elongation......Page 596
Remarks......Page 600
Analysis of RNA released after transcription......Page 601
Acknowledgment......Page 602
Probing Chromatin Immunoprecipitates with CpG-Island Microarrays to Identify Genomic Sites Occupied by DNA-Binding Proteins......Page 603
Chromatin Immunoprecipitation......Page 606
Day 116......Page 607
Day 2......Page 609
Step 1......Page 610
Step 12......Page 611
REAGENTS......Page 612
Day 1......Page 613
Day 2......Page 614
Step 1......Page 616
Step 4......Page 617
Reagents......Page 618
Analysis......Page 619
Conclusions......Page 621
Acknowledgments......Page 622
Isolation of RNA Polymerase Suppressors of a (p)ppGpp Deficiency......Page 623
Selection I: Direct Selection for Mutants Restoring Growth on Minimal Medium......Page 624
Selection III: Rifampicin-Resistant Mutants Screened as M+......Page 625
Genetic Linkage by P1 Transduction......Page 626
Sequence Localization of Mutations......Page 627
Comments......Page 628
Strategy and Practical Considerations......Page 629
Repressor Activity In Vivo......Page 631
Regulation of Repression in Response to Specific Signals......Page 632
Functional Relationships to Corepressors and Components of the Transcriptional Apparatus......Page 633
LexA fusion plasmids......Page 634
Stock Solutions......Page 635
Assays of b-Galactosidase Activity......Page 637
Reagents......Page 638
Western Blot Analysis......Page 640
Concluding Remarks......Page 641
Random......Page 642
Targeted......Page 647
Selections......Page 649
Methods......Page 651
Concluding Remarks and Recommendations......Page 653
Acknowledgment......Page 656