There and Back Again: A Structural Atlas of RNAP |
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Part I From Promoter Recognition to Promoter Escape |
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Chapter 1 Where it all Begins: An Overview of Promoter Recognition and Open Complex Formation |
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Stephen Busby, Annie Kolb and Henri Buc |
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1.1 Gene Expression as a Driver of Life |
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1.2 Escherichia coli RNA Polymerase |
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1.3 Promoters and Core Promoter Elements |
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1.4 Biochemistry: It Works with RNA Polymerase! |
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1.5 Biochemistry of Promoter Regulation |
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1.6 A Word about the Intracellular Environment |
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1.7 Coupling Transcription to Changes in a Complex Environment |
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1.8 A Global View of the RNA Polymerase Economy |
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1.9 The Real World, Emergency Procedures and RNA Polymerase |
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1.10 This is just the Beginning! |
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Chapter 2 Opening the DNA at the Promoter; The Energetic Challenge |
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2.2 Structural Characterization |
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2.2.1 Crystal Structure of the Holoenzyme |
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2.2.2 Crystal Structure of the Holoenzyme with Fork junction DNA |
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2.2.3 Structural Model of the Open Complex |
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2.3 Physical Characterization and Structure of the Intermediates |
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2.4 Finding the Promoter. Induced fit and Indirect Sequence Recognition |
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2.5 Formation of the Closed Complex |
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2.5.1 On a Unique Structure of the Closed Complex |
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2.5.2 Role of Upstream Contacts for the Stability of the Closed Complex and in Leading the Complex Towards Subsequent Isomerization |
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2.6 The First Isomerization Step. The role of Sigma, Formation of Specific Interactions |
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2.6.2 Nucleation of the Single Stranded Region and its Propagation |
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2.6.3 Phasing of 10 and 35 Regions, the Role of the Spacer |
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2.6.4 Probing Possible Sequential Linear Pathways by the use of Temperature |
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2.6.5 Specific Protein Domains Destabilize the Intermediates in the Pathway |
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2.6.6 Overstabilization is sometimes used as a Regulatory Mechanism |
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2.7 Formation of Transcriptionally Active Open Complex and the Rate-limiting Step: Protein Conformational Changes or DNA Melting? |
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2.8 A Rugged Energy Landscape |
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2.9 Summary and Conclusions |
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Chapter 3 Intrinsic In vivo Modulators: Negative Supercoiling and the Constituents of the Bacterial Nucleoid |
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Georgi Muskhelishvili and Andrew Travers |
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3.2 DNA Superhelicity Structures and Implications |
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3.3 Structure of the Bacterial Nucleoid |
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3.4 Supercoiling Utilization |
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3.5 Promoter Structure and DNA Supercoiling |
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3.6 Role of RNA Polymerase Composition |
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3.6.1 Exchange of σ Factors |
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3.7 Model and Implications |
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3.9 Cooperation with Nucleoid Associated Proteins |
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3.10 Conversion of Supercoil Energy into Genomic Transcript Patterns |
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Chapter 4 Transcription by RNA Polymerases: From Initiation to Elongation, Translocation and Strand Separation |
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4.2 Transition from the Initiation to the Elongation Phase |
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4.2.2 Multi-subunit RNA Polymerases |
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4.3 Translocation and Strand Separation |
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4.3.2 Multi-subunit Cellular RNAPs |
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4.4 Additional Similarities between Single and Multi-subunit Polymerases |
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Chapter 5 Single-molecule FRET Analysis of the Path from Transcription Initiation to Elongation |
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Achillefs N. Kapanidis and Shimon Weiss |
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5.2 Methodology: FRET and ALEX Spectroscopy |
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5.3 Transcription Mechanisms Addressed using Single-molecule FRET and ALEX |
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5.4 Fate of Initiation Factor 670 in Elongation |
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5.5 Mechanism of Initial Transcription |
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5.6 Kinetic Analysis of Initial Transcription and Promoter Escape |
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5.7 Comparison of FRET Approaches with Magnetic-trap Approaches |
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Chapter 6 Real-time Detection of DNA Unwinding by Escherichia coli RNAP: From Transcription Initiation to Termination |
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Terence R. Strick and Andrey Revyakin |
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6.2 Twist Deformations at the Promoter |
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6.3 Magnetic Trapping and Supercoiling of a Single DNA Molecule |
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6.3.1 General Features of the Magnetic Trap |
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6.3.2 Calibrating the DNA Sensor |
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6.4 Characterization of RPo at two Canonical Promoters |
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6.4.1 Structural Characterization of RPo |
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6.4.2 Kinetic Analysis of RPo |
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6.4.3 Effect of Environmental Variables on Kinetics of RPo |
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6.5 Promoter Escape by DNA Scrunching |
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6.5.1 Characterization of DNA Scrunching during Abortive Initiation |
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6.5.2 Characterization of DNA Scrunching during Promoter Escape |
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Part II Transcription Elongation and Termination |
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Interlude: The Engine and the Brake |
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Henri Buc and Terence Strick |
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I.2.1 Mechano-chemical Coupling at the Catalytic Site |
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I.2.2 Coupling between Translocation and Topology |
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Chapter 7 Substrate Loading, Nucleotide Addition, and Translocation by RNA Polymerase |
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Jinwei Zhang and Robert Landick |
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7.1 Basic Mechanisms of Transcript Elongation by RNA Polymerase |
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7.1.1 Active-site Features of an Elongation Complex |
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7.1.2 The Nucleotide Addition Cycle |
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7.1.3 Pyrophosphorolysis and Transcript Cleavage |
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7.1.4 Regulation of Transcript Elongation by Pauses |
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7.2 Structural Basis of NTP Loading and Nucleotide Addition |
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7.2.1 Bridge-helix-centric Models of Nucleotide Addition and Translocation |
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7.2.2 Central Role of the Trigger Loop in Nucleotide Addition and Pausing |
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7.2.3 A Trigger-loop Centric Mechanism for Substrate Loading and Catalysis |
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7.3 Models of Translocation: Power-stroke versus Brownian Ratchet |
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7.3.1 Key Distinctions between Power-stroke and Brownian Ratchet Models |
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7.3.2 Power-stroke Models |
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7.3.3 Brownian Ratchet Models |
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7.3.4 Technical Outlook in Detecting the Precise Translocation Register |
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7.4 Kinetic Models of Nucleotide Addition |
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7.4.1 Allosteric NTP Binding Model |
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7.4.2 NTP-driven Translocation Model |
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7.4.3 Two-pawl Ratchet Model |
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7.4.4 Biophysical Models for Transcript Elongation |
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7.5 Technological Advances in Studies of Transcript Elongation |
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Chapter 8 Regulation of RNA Polymerase through its Active Center |
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Sergei Nechaev, Nikolay Zenkin and Konstantin Severinov |
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8.2 Regulatory Checkpoints of the RNAP Active Center |
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8.2.1 Versatility of the Active Center. How many Metals are Enough? |
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8.2.2 Delivery of NTPs to the Active Center. How many Channels are Enough? |
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8.2.3 Nucleotide Selection. How many Steps are Enough? |
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8.3 Regulators that Target the RNAP Active Center |
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8.3.1 Small-molecule Effectors of RNAP |
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8.3.2 Regulation of RNAP by Proteins that Bind in the Secondary Channel |
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8.4 Transcript Proofreading |
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8.4.1 Transcriptional Proofreading through Pyrophosphorolysis |
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8.4.2 Proofreading by Transcript Cleavage Factors |
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8.4.3 Transcript-assisted Proofreading. A New Class of Ribozymes? |
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Chapter 9 Kinetic Modeling of Transcription Elongation |
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Lu Bai, Alla Shundrovsky and Michelle D. Wang |
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9.3 Mechano-chemical Coupling of Transcription |
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9.3.1 NTP Incorporation Cycle |
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9.3.2 NTP Incorporation Pathway in a Simple Brownian Ratchet Model |
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9.3.3 NTP Incorporation Pathways in more Elaborate Brownian Ratchet Models |
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9.3.4 NTP Incorporation Pathway in a Power-stroke Model |
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9.3.5 Elongation Kinetics |
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9.3.6 Force-dependent Elongation Kinetics |
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9.4 Sequence-dependent RNAP Kinetics |
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9.4.1 Thermodynamic Analysis of the TEC |
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9.4.2 Sequence-dependent NTP Incorporation Kinetics in Brownian Ratchet Models |
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9.4.3 Model Predictions of Pause Locations, Kinetics and Mechanisms |
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Chapter 10 Mechanics of Transcription Termination |
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10.2 Structure/Function Overview of the Elongation Complex (EC) |
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10.3 Mechanism of Intrinsic Termination |
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10.3.2 The Termination Phase |
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10.4 Mechanism of Rho Termination |
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Conclusion Past, Present, and Future of Single-molecule Studies of Transcription |
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Carlos Bustamante and Jeffrey R. Moffitt |
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C.2 RNA Polymerase as a Molecular Machine: Past and Present |
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C.3 Technical Developments in Optical Tweezers |
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C.4 A Look into the Future |
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Subject Index |
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