RNA Polymerase and Associated Factors Part C

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Construction of E. coli Strains with Chromosomal Gene Fusions to the rpoC Gene......Page 33
Generating Linear DNA Fragments That Carry gfpuv, cfp, or yfp Genes and the bla Gene Flanked by Chromosomal Sequences Near th......Page 35
Purification of RNAP Tagged with Green, Yellow, or Cyan Fluorescent Proteins......Page 36
Physical and Biochemical Properties of RNAP Tagged with Green, Yellow, or Cyan Fluorescent Proteins......Page 37
Potential Uses of RNAP Tagged with Green, Yellow, or Cyan Fluorescent Proteins......Page 39
Acknowledgments......Page 40
Introduction......Page 41
General Considerations......Page 43
Method 1. Purification of His-Tagged RNAP Core Enzyme......Page 45
Lysis......Page 46
Polymin P Fractionation......Page 47
A1.5M Sizing Chromatography......Page 48
Method 3. Purification of RNAP Using Heparin-Sepharose, Sephacryl S-300, and Mono-Q......Page 49
Method 5. Core RNA Polymerase Lacking d (E) and Purified d Protein......Page 50
Method 6. Overexpression and Purification of d......Page 51
Method 7. Purification of s Factors......Page 52
Purification of sD......Page 53
Method 8. Reconstitution of Holoenzyme......Page 54
Acknowledgments......Page 55
Determination of Escherichia coli RNA Polymerase Structure by Single Particle Cryoelectron Microscopy......Page 56
Purification of Escherichia coli Core RNA Polymerase......Page 58
Core RNA Polymerase Catalytic Activity Assay......Page 60
Preparation of Holey Carbon Grids......Page 61
Cryo-EM of RNA Polymerase......Page 62
Digitization of Images......Page 63
Correction of the Contrast Transfer Function......Page 65
Reference-Free Alignment and Multireference Alignment......Page 67
Euler Angle Assignment......Page 68
Resolution Estimation and Filtering of the Final Three-Dimensional Reconstruction......Page 69
Fitting of the RNAP Crystal Structure from T. aquaticus......Page 71
Homology Modeling of the E. coli RNA Polymerase......Page 72
Concluding Remarks......Page 73
Acknowledgments......Page 74
Crystallographic Analysis of Thermus aquaticus RNA Polymerase Holoenzyme and a Holoenzyme/Promoter DNA Complex......Page 75
Core Enzyme......Page 76
sA......Page 78
DNA......Page 79
Holoenzyme/Fork-Junction DNA Complex......Page 80
Holoenzyme......Page 81
Phasing, Model Building, and Refinement......Page 82
Concluding Remarks......Page 85
Acknowledgments......Page 86
Purification of Rhodobacter sphaeroides RNA Polymerase and Its Sigma Factors......Page 87
Purification of RNA Polymerase......Page 88
Affinity Purification of R. sphaeroides Core RNA Polymerase......Page 89
Highly Purified R. sphaeroides RNA Polymerase......Page 90
Purification of Recombinant R. sphaeroides RpoD......Page 92
Purification of Recombinant R. sphaeroides RpoH1 and RpoHII......Page 93
Purification of Recombinant R. sphaeroides RpoE......Page 94
Purification of MBP-ChrR......Page 95
Assay Conditions......Page 96
Acknowledgments......Page 98
In Vitro Transcription Assays Using Components from Methanothermobacter thermautotrophicus......Page 99
Growth of Methanothermobacter thermautotrophicus......Page 100
Purification of M. thermautotrophicus RNA Polymerase......Page 101
Promoter-Specific in Vitro Transcription......Page 103
Purification and Use of Stalled Transcription Ternary Complexes to Assay Transcription Elongation......Page 104
Acknowledgments......Page 105
Isolation and Characterization of Streptomyces coelicolor RNA Polymerase, Its Sigma, and Antisigma Factors......Page 106
Preparation of Cell Extracts from Streptomyces coelicolor Mycelia......Page 108
Polyethyleneimine (PEI) Fractionation......Page 109
Mono Q Column......Page 110
Reconstitution Assay......Page 111
In Vitro Transcription Assay......Page 112
Sigma and Antisigma Interaction Analyzed by Native PAGE......Page 113
Sigma and Antisigma Interaction Analyzed by Surface Plasmon Resonance......Page 114
Acknowledgments......Page 115
Bacteriophage N4-Coded, Virion-Encapsulated DNA-Dependent RNA Polymerase......Page 116
Buffers......Page 119
Step 3. Virion Disruption......Page 120
Step 7. SP Chromatography and Mono Q Concentration......Page 121
Step 2. Co2+ Metal Affinity Chromatography......Page 122
Step 1. Protein Overproduction and Cell Lysis......Page 123
Purification of Untagged Mini-vRNAP by DNA Affinity Chromatography......Page 124
Step 2. Protein Overproduction and Cell Lysis......Page 125
Concluding Remarks......Page 126
Acknowledgment......Page 127
Preparation and Characterization of Recombinant Thermus aquaticus RNA Polymerase......Page 128
Plasmids Expressing Taq rpo Genes......Page 130
Taq RNAP sA Subunit......Page 133
In Vitro Taq RNAP Reconstitution......Page 136
Preparation of Taq RNAP from E. coli Cells Cooverexpressing Taq rpo Genes......Page 137
In Vitro Transcription......Page 138
Site-Specific Mutagenesis......Page 140
Acknowledgments......Page 142
Purification and Assay of Upstream Activation Factor, Core Factor, Rrn3p, and Yeast RNA Polymerase I......Page 143
General Methods......Page 144
Purification of Pol I......Page 146
Purification of CF......Page 147
Purification of UAF......Page 148
Solutions......Page 149
Solutions......Page 150
Solutions......Page 151
Pol I Transcription Assay......Page 153
Concluding Remarks......Page 154
Purification and Transcriptional Analysis of RNA Polymerase I Holoenzymes from Broccoli (Brassica oleracea) and Frog (Xenopus......Page 155
Crude Nuclear Extract Preparation......Page 157
Pol I Holoenzyme Purification......Page 159
Promoter-Independent (Nonspecific) RNA Polymerase I Activity Assay......Page 163
Promoter-Dependent Pol I Transcription and Detection of Transcripts......Page 166
Purification of Xenopus laevis Pol I Holoenzyme Activity......Page 168
Procedure......Page 169
Activities Associated with Brassica and Xenopus Pol I Holoenzymes......Page 171
Assays and Affinity Purification of Biotinylated and Nonbiotinylated Forms of Double-Tagged Core RNA Polymerase II from Sacch......Page 173
Materials and Reagents......Page 174
Buffers......Page 175
GST-BirA Expression and Purification......Page 176
Yeast Whole Cell Lysate Preparation......Page 177
Activity Assay for Histidine-Tagged Pol II......Page 178
Binding Capacity of Ni2+-NTA Agarose for Pol II......Page 179
Biotinylation of Double-Tagged Pol II......Page 182
Affinity Purification of Biotinylated Pol II......Page 185
Purification of Nonbiotinylated Pol II Core Enzyme......Page 186
Binding Capacity of Ni2+-NTA Agarose for Purified Pol II......Page 187
Subunit Composition of Affinity-Purified Pol II......Page 188
Acknowledgments......Page 190
Dephosphorylation of the Carboxyl-Terminal Domain of RNA Polymerase II......Page 191
Consideration of Substrate......Page 192
Dephosphorylation of Synthetic CTD Phosphopeptides......Page 193
rCTD......Page 194
RNAP IIO......Page 195
Dephosphorylation of Purified Calf Thymus RNAP IIO......Page 196
Dephosphorylation of RNAP IIO Contained in Nuclear Extracts......Page 197
Dephosphorylation of RNAP IIO in Elongation Complexes......Page 198
Acknowledgments......Page 201
Strains and Media......Page 202
Method I. Preparation of Fission Yeast Extracts......Page 203
Method II. Construction of TspRI-Generated 9 Nucleotide 3' Overhang (i.e., 3'-Tailed Templates)......Page 204
Promoter-Dependent Transcription Reaction and RNA Purification......Page 206
Method IV. Purification of Double Epitope-Tagged S. pombe RNA Polymerase III Affinity Purification......Page 207
Glycerol Gradient Sedimentation......Page 208
Discussion......Page 210
Purification of Highly-Active and Soluble Escherichia coli s70 Polypeptide Overproduced at Low Temperature......Page 211
Cell Growth and Induction......Page 212
Purification Procedure......Page 213
In Vitro Transcription Assay for Sigma Activity......Page 215
Acknowledgments......Page 217
Expression, Purification of, and Monoclonal Antibodies to s Factors from Escherichia coli......Page 218
Purification Notes......Page 222
Purification of sE......Page 223
Purification Notes......Page 225
Purification of sFecI......Page 226
Production of s-Specific Monoclonal Antibodies......Page 228
Acknowledgments......Page 229
Introduction to Protein-Binding Studies......Page 230
Introduction to Fluorescence Labeling of Proteins......Page 232
Overproduction of HMK-His6-b'(100-309) and Purification of Inclusion Bodies......Page 234
Procedure......Page 235
Procedure......Page 236
Electrophoretic Mobility Shift Assay for Complex Formation of Labeled s70 and b' Fragment......Page 237
Comments......Page 239
LRET Assay for Protein-Protein Interaction of Labeled s70 and b' Fragment......Page 240
Comments and Variations......Page 242
Assay of Escherichia coli RNA Polymerase: Sigma-Core Interactions......Page 244
Plasmids and DNA Manipulations......Page 245
Overproduction and Purification of Fusion Proteins......Page 247
GST Pull-Down Experiments......Page 248
Initial Interface......Page 249
Acknowledgments......Page 250
Purification and Characterization of Bacteriophage-Encoded Inhibitors of Host RNA Polymerase: T-Odd Phage gp2-like Proteins......Page 251
Overexpression and Purification of Recombinant T7 gp2......Page 252
Protocol......Page 253
Overexpression and Purification of RNAP Subunits or Their Fragments......Page 254
Protocol......Page 255
Protocol......Page 256
Protocol......Page 258
Establishing the Presence of Host RNAP Inhibitor in Phage-Infected Cells......Page 260
Protocol......Page 261
Protocol......Page 262
Notes......Page 263
Acknowledgments......Page 264
Motif Redundancy Measured by the Reduction in Shannon Entropy......Page 265
Functional Ranking of Individual Sequences......Page 268
Sequence Analysis with Neural Networks......Page 271
Ranking Function Preprocessing for Neural Networks......Page 272
Computational Detection of Vertebrate RNA Polymerase II Promoters......Page 274
Dragon Promoter Finder......Page 277
Dragon Promoter Finder 1.3 Model......Page 278
System Training and Tuning......Page 281
Model Testing......Page 282
Concluding Remarks......Page 286
Detection of DNA-Binding Helix-Turn-Helix Motifs in Proteins Using the Pattern Dictionary Method......Page 288
Consensus Methods......Page 291
Profile Methods......Page 292
Pattern Discovery Methods......Page 293
Pattern Dictionary Method......Page 294
Detection Phase......Page 296
Output of Algorithm......Page 297
Analysis of Families of DNA-Binding Proteins......Page 298
Web Page......Page 301
Acknowledgments......Page 302
DNA Microarrays and Bacterial Gene Expression......Page 303
PCR versus Long Oligonucleotide Arrays......Page 305
Protocol for Printing Slides (PCR Based or Long Oligonucleotides)......Page 306
Verification of Array Quality......Page 307
Experimental Design......Page 308
Growth of Cells and RNA Preparation......Page 309
Labeling: Indirect Incorporation Using Aminoallyl dUTP......Page 310
Hybridization of Probe to Microarrays Slides......Page 313
Normalization......Page 314
Identification of Differentially Expressed Genes......Page 315
Data Storage......Page 316
Acknowledgments......Page 317
Analysis of Microarray Data for the marA, soxS, and rob Regulons of Escherichia coli......Page 318
Purification and Activity Assays of RapA, the RNA Polymerase-Associated Homolog of the SWI/SNF Protein Superfamily......Page 321
Purification of RapA from RapA-RNAP Holoenzyme Complex......Page 322
In Vitro Transcription......Page 326
ATPse Assay......Page 328
Escherichia coli Proteins Eluted from Mono Q Chromatography, a Final Step During RNA Polymerase Purification Procedure......Page 329
Cell Disruption......Page 336
Single-Stranded DNA-Agarose Chromatography......Page 337
Mono Q Chromatography......Page 338
Techniques for Studying the Oxygen-Sensitive Transcription Factor FNR from Escherichia coli......Page 339
Growth of Cells......Page 342
Preparation of Cell Lysate......Page 343
Protein Purification......Page 344
Protein Purification Equipment......Page 345
Anaerobic Procedures......Page 346
FNR* Mutants......Page 347
Solid Media......Page 348
Sparging Station......Page 349
Acknowledgments......Page 351
Assay of Transcription Modulation by Spo0A of Bacillus subtilis......Page 352
Purification of Spo0A......Page 353
Purification of Spo0ABD......Page 354
Activation of Spo0A......Page 355
Protocol......Page 357
Notes......Page 358
Protocol......Page 359
Notes......Page 360
Protocol......Page 361
Rationale......Page 362
Acknowledgments......Page 363
Assay of Prokaryotic Enhancer Activity over a Distance In Vitro......Page 364
Materials......Page 365
Purification of DNA, Proteins, and Protein Complexes......Page 366
Purification of E. coli Core RNAP......Page 367
Expression and Purification of Recombinant E.coli NtrB......Page 368
Single- and Multiple-Round Assays for Enhancer-Dependent Transcription......Page 370
Analysis of the Rate of Enhancer-Promoter Communication......Page 374
Purification of Initiation and Early Elongation Complexes by Gel Filtration......Page 376
Acknowledgment......Page 377
Functions of DnaA......Page 378
Structure of DnaA......Page 379
Different DNA-Binding Properties of DnaA......Page 380
Experimental Protocol......Page 381
Purification of E. coli DnaA......Page 383
Experimental Protocol......Page 384
Specific Binding of DnaA to DNA......Page 385
Experimental Protocol......Page 386
DNase l Footprinting......Page 387
In Vitro Transcription......Page 388
Experimental Protocol......Page 389
Analysis of Transcription Factor Interactions at Sedimentation Equilibrium......Page 390
Overview of the Sedimentation Equilibrium Method......Page 391
Self-Association......Page 392
Heteroassociation......Page 396
Noninteracting components......Page 399
Thermodynamic Linkage......Page 400
Temperature Dependence of Association......Page 401
Volume......Page 402
Sample Concentration......Page 403
Buffer......Page 404
Rotor Speed and Duration of Run......Page 405
Determination of Partial Specific Volume......Page 406
Data Analysis......Page 408
Prospects......Page 409
Acknowledgments......Page 410
Single-Molecule Studies of DNA Architectural Changes Induced by Regulatory Proteins......Page 411
Preparation of Microscope Flow Chamber......Page 412
Cleaning......Page 413
Flow Chambers......Page 414
Surface Density......Page 415
Beads......Page 416
Data Recording......Page 418
Particle Tracking......Page 419
Assay of an Intrinsic Acetyltransferase Activity of
the Transcriptional Coactivator CIITA......Page 421
Preparation of Recombinant CIITA from Baculovirus......Page 422
Transfection of Sf9 Cells to Generate Viral Seed Culture......Page 423
Preparing a Large-Scale Viral Stock......Page 424
Protein Immunoprecipitation......Page 425
Immunoprecipitation of CIITA from Transfected Hela Cells......Page 426
Western Immunoblot Analysis of CIITA......Page 427
Histone Acetyltransferase Assay......Page 428
Purification and Assay of Saccharomyces
cerevisiae Phosphatase That Acts on the C-Terminal
Domain of the Largest Subunit of......Page 430
RNA Polymerase II as Substrate......Page 431
Overexpressed Protein from Bacterial Cells......Page 433
Recombinant Protein from Baculovirus-Infected Cells......Page 434
Native Protein from Yeast Cells Using the Tandem Affinity Purification Procedure......Page 436
Native Protein from Yeast Cells......Page 438
The Transcriptional Response to Cyclic AMP......Page 440
A Family of Transcriptional Regulators......Page 441
Phosphorylation-Dependent Activation......Page 442
Activation of Various Signaling Pathways Lead to CREB Phosphorylation......Page 444
Riboprobes......Page 445
Characterizing CREB Kinases......Page 446
ACT......Page 448
Expression and Purification of CREB and CREM Proteins......Page 449
Preparation, Labeling, and Purification of the Antisense Riboprobe......Page 452
RNase Digestion and Purification......Page 453
Gel......Page 454
From Transfected Cells in Culture......Page 455
From Mouse Tissue......Page 456
Solutions......Page 457
Quantification of Results......Page 458
Acknowledgments......Page 459
Functional Analysis of TFIID Components Using Conditional Mutants......Page 460
Characterization of Growth of Temperature-Sensitive TAF Mutants......Page 461
Plating and Recovery Assay......Page 463
Liquid Growth Curves......Page 464
TAF Inactivation Protocol......Page 465
Isolation of Total RNA and Measurement of Total Poly(A)+ mRNA......Page 467
Analyzing Induced Gene Expression in Temperature-Sensitive Mutants......Page 468
Preparation of Mini Whole Cell Extract for Western Blotting......Page 469
Materials and Reagents......Page 470
Depletion Methodology......Page 473
Immunoaffinity Purification of Mammalian Protein Complexes......Page 476
Generation of Epitope-Tagged Protein-Expressing Cells......Page 478
Transfection of Retroviral Vector into Packaging Cells......Page 480
Retroviral Transduction......Page 481
Sorting of Transduced Cells......Page 482
Detection of Expression of the Epitope-Tagged Protein......Page 484
Growth of cells......Page 486
Materials......Page 487
Procedure......Page 488
Anti-HA Antibody Immunoaffinity Purification......Page 489
Acknowledgment......Page 490
Interaction of Gal4p with Components of
Transcription Machinery In Vivo......Page 491
Formaldehyde-Based In Vivo Cross-Linking and ChIP Assay......Page 492
DNA Substrate......Page 494
Formaldehyde Concentration and Cross-Linking Time......Page 495
Protocol for Formaldehyde-Based In Vivo Cross-Linking and ChIP Assay......Page 496
Other Considerations......Page 499
Acknowledgments......Page 500
Dominant-Negative Mutants of Helix-Loop-Helix
Proteins: Transcriptional Inhibition......Page 501
Structure of B-HLH Transcription Factors......Page 503
DNA Binding Stabilizes B-HLH-ZIP Dimers......Page 506
Design of the Acidic Extension......Page 508
A-HLH DNs Inhibit DNA Binding of B-HLH Proteins in a Dimerization-Specific Manner......Page 510
Biological Uses of Dominant Negatives......Page 511
Purification and Transcription Repression by Negative Cofactor 2......Page 514
Solutions......Page 515
Procedure......Page 516
Procedure for EMSA......Page 517
Solutions......Page 518
Procedure......Page 519
Coupling of Antibody to Beads......Page 520
Detection of NC2 on Genes by Chromatin IP8,9......Page 521
Procedure......Page 522
Repression of Transcription by NC2......Page 524
Procedure......Page 525
Acknowledgments......Page 526
Hap1p Photofootprinting as an In Vivo Assay of Repression Mechanism in Saccharomyces cerevisiae......Page 527
Sample Preparation......Page 529
Primer Extension Mapping......Page 532
Recipes......Page 533
Troubleshooting......Page 535
Analysis of Activator-Dependent Transcription Reinitiation In Vitro......Page 536
General Outline of the Experimental Strategy......Page 538
RNA Quantification by Comparison to Reference RNA......Page 539
Transcription System......Page 541
Solid-Phase Transcription......Page 542
In Vitro Transcription and Determination of Reinitiation Rate......Page 544
RNA Purification and Primer Extension Analysis......Page 545
Analysis of Proteins Assembled on the Template......Page 547
Activation of Reinitiation by Heat Shock Factor......Page 548
Other Methodologies for Studying Reinitiation......Page 550
Molecular Analysis of Activator Engagement with RNA Polymerase......Page 551
SR4 from Escherichia coli s70......Page 552
SR4 from Thermotoga maritima sigA......Page 553
T4 Bacteriophage AsiA......Page 554
Analytical Ultracentrifugation of AsiA......Page 555
Sedimentation Velocity......Page 556
Sedimentation Equilibrium......Page 559
Protein-Protein Cross-Linking......Page 560
Nuclear Magnetic Resonance Spectroscopy of Activator/RNAP Complexes......Page 562
NMR "Footprinting"......Page 563
Interfacial NMR......Page 564
Acknowledgment......Page 567
Purification and Protein Interaction Assays of the VP16C Transcription Activation Domain......Page 568
Rationale......Page 570
Methods......Page 571
Rationale......Page 572
Methods......Page 573
Rationale......Page 574
Methods......Page 575
Results......Page 577
Rationale......Page 578
Methods......Page 579
Acknowledgments......Page 581
Rapid Quench Mixing to Quantify Kinetics of Steps in Association of Escherichia coli RNA Polymerase with Promoter DNA......Page 582
Es70 RNA Polymerase......Page 584
Rapid Quench Association Kinetics Experiments......Page 585
Data Analysis to Obtain Observed Rate Constants and Fractional Occupancy of Promoter DNA......Page 587
Kinetic Background......Page 589
Concluding Remarks......Page 591
Acknowledgments......Page 593
Determination of RNA Polymerase Binding and Isomerization Parameters by Measuring Abortive Initiations......Page 594
Data Processing......Page 595
Fluorescence-based Abortive Initiation Assays......Page 598
Caveat......Page 600
Probing the Role of Region 2 of Escherichia coli s70 in Nucleation and Maintenance of the Single-Stranded DNA Bubble i......Page 602
RNAP-Promoter Interactions......Page 603
Sigma Factor Region 2.3 and Nucleation of DNA Strand Separation......Page 605
Use of Forked DNAs to Dissect Steps in Formation of Open Complexes......Page 606
Experimental Procedures......Page 607
Reagents and Techniques......Page 608
Interpretation of Data......Page 615
Acknowledgments......Page 616
On the Use of 2-Aminopurine as a Probe for Base Pair Opening During Transcription Initiation......Page 617
Methods for Preparing 2-Aminopurine Containing Bacterial Promoters......Page 618
Determination of Base Pair Opening Rates......Page 620
Equipment......Page 621
Data Analysis......Page 622
Promoter Clearance Assay......Page 624
Concluding Remarks......Page 625
Single-Molecule DNA Nanomanipulation: Detection of Promoter-Unwinding Events by RNA Polymerase......Page 626
Methods and Protocols......Page 630
Preparing a Hydrophobic Surface by Silanization......Page 631
Assembly of Capillary into Flow Cell......Page 632
Materials......Page 633
Preparation of 1-kb Labeled DNA Fragments......Page 634
Materials......Page 636
Materials......Page 637
Calibration of Bead Image......Page 638
Force Calibration......Page 640
Force Calibrations Based on Stretching Transitions in Supercoiled DNA......Page 641
Materials......Page 642
Temporal Analysis of Unwinding Signal......Page 643
Signal-to-Noise Analysis......Page 644
Detecting DNA Bending/Compaction......Page 645
Concluding Remarks......Page 646
Acknowledgments......Page 647
Simple Fluorescence Assays Probing Conformational Changes of Escherichia coli RNA Polymerase During Transcription Initiation......Page 648
Fluorescence Spectroscopy......Page 649
Fluorescence Quenching......Page 651
Tryptophan Fluorescence of RNA Polymerase as a Reporter of the Existence of Multiple Intermediates in the Pathway of Open Compl......Page 652
A Fluorescent Substrate, Tb-GTP, as a Probe for Active Site Conformation of RNAP during RPc to RPo Conversion......Page 653
Concluding Remarks......Page 655
Measuring Control of Transcription Initiation by Changing Concentrations of Nucleotides and Their Derivatives......Page 656
Stepwise Procedure for Measuring Effects of ppGpp or iNTPs on Transcription Initiation......Page 657
Reagents......Page 658
Rationale......Page 659
Other Comments......Page 660
Stepwise Procedure......Page 661
Other Comments......Page 662
Stepwise Procedure......Page 663
Other Comments......Page 664
Stepwise Procedure......Page 665
Concluding Remarks......Page 667
Reagents and Enzymes......Page 668
Plasmids......Page 669
Preparation of Reagents......Page 672
Procedure for setting up Reactions......Page 673
5' Mapping of Transcripts......Page 675
DNA Sequencing......Page 676
Transcription from Promoters on Plasmid......Page 677
Mapping PA Orientation......Page 679
Mapping 5' Start Sites of PA and PC......Page 681
Deletion of T7 and attB’OB Regions......Page 684
Delection of the attP’OP Region......Page 686
Effect of IHF on PB Transcript......Page 692
RpoC Terminator is Bidirectional......Page 693
Determination of the 3' End of Transcripts......Page 695
Enhancer-Dependent Transcription by Bacterial RNA Polymerase: The b Subunit Downstream Lobe Is Used by s54 During Op......Page 696
Overexpression and Purification of Recombinant s54......Page 697
Overexpression, Purification, and In Vivo Reconstitution of Hexahistidine-Tagged Wild-Type and b(d186-433) Cor......Page 698
Overexpression and Purification of 6His-Tagged E. coli s54 Activator Phage Shock Response Protein F (PspF)......Page 700
Promoter Complex Formation by s54-RNAP......Page 702
In Vitro Transcription Assays......Page 705
Concluding Remarks......Page 707
Mutational Analysis and Structure of the Phage SP6 Promoter......Page 708
Saturation Mutagenesis of the SP6 Promoter......Page 709
Construction of a Two-Vector System......Page 711
Determination of Plasmid Copy Number......Page 712
Determination of In Vivo Promoter Activity......Page 713
Relative Promoter Strength in Vitro......Page 714
Purification of SP6 RNA Polymerase......Page 715
Graphic Presentation of Saturation Mutagenesis Data......Page 716
Acknowledgments......Page 718
Fluorescence Methods for Studying the Kinetics and Thermodynamics of Transcription Initiation......Page 719
2-Aminopurine as a Probe for RNAP-DNA Interactions......Page 721
Equilibrium Dissociation Constant......Page 724
Association Rate Constant......Page 728
Kinetics of Open Complex Formation in the Absence of Initiating Nucleotide......Page 730
Kinetics of Open Complex Formation in the Presence of Initiating Nucleotide......Page 732
Kinetics of NTP Binding during Initiation......Page 733
Kinetics of Initial RNA Synthesis......Page 734
Acknowledgment......Page 737
In Vitro Studies of the Early Steps of RNA Synthesis by Human RNA Polymerase II......Page 738
Theory......Page 739
Method......Page 740
Theory......Page 743
Method......Page 745
Theory......Page 746
Method......Page 747
Interpretation of Results......Page 748
Theory......Page 749
Method......Page 750
Method......Page 751
Interpretation of Results......Page 752
Site-Specific Protein-DNA Photocross-Linking of Purified Complexes: Topology of the RNA Polymerase II Transcription Initiatio......Page 753
Overview of the Procedure......Page 754
General Considerations......Page 755
Enzymatic Synthesis of Photoprobes......Page 756
Precautions......Page 759
Preparation of Polyacrylamide:Bac Gels......Page 760
UV Irradiation of Gels......Page 761
Nuclease Digestion and SDS-PAGE Analysis......Page 762
Analysis of RNA Polymerase II Preinitiation Complex......Page 763
Acknowledgments......Page 764
Assay of Promoter Melting and Extension of mRNA: Role of TFIIH Subunits......Page 765
Preparation of Human Whole Cell Extracts (WCE)......Page 768
Preparation of Baculovirus-Infected Insect Cell Extracts......Page 769
Purification......Page 770
Principle......Page 772
Transcription Reaction with WCE......Page 773
Reconstituted Transcription System......Page 774
Production of Heteroduplex......Page 776
Assay......Page 777
Promoter Opening and Permanganate Assay......Page 779
Assay......Page 780
Promoter Escape......Page 781
Analysis of Results......Page 782
Conclusion......Page 784
Acknowledgments......Page 785
Assays for Investigating the Mechanism of Promoter Escape by RNA Polymerase II......Page 786
Materials......Page 787
Reconstitution of Promoter-Specific Transcription by RNA Polymerase II and General Initiation Factors......Page 788
Use of Dinucleotide-Primed Transcription Assays to Investigate the Mechanism of Promoter Escape by RNA Polymerase II......Page 789
Preparation and Analysis of Early RNA Polymerase II Elongation Intermediates......Page 790
Use of Bubble DNA Templates to Investigate the Mechanism of Promoter Escape by RNA Polymerase II......Page 792
Acknowledgments......Page 793