RNA Polymerases as Molecular Motors
By: Prof. Stephen Neidle (Editor), Prof. Henri Buc (Editor), Bianca Sclavi (Contribution by), Terence Strick (Editor), Andrew Travers (Contribution by)
Hardcover | 1 April 2009
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331 Pages
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The cell can be viewed as a 'collection of protein machines' and understanding these molecular machines requires sophisticated cooperation between cell biologists, geneticists, enzymologists, crystallographers, chemists and physicists. To observe these machines in action, researchers have developed entirely new methodologies for the detection and the nanomanipulation of single molecules. This book, written by expert scientists in the field, analyses how these diverse fields of research interact on a specific example - RNA polymerase. The book concentrates on RNA polymerases because they play a central role among all the other machines operating in the cell and are the target of a wide range of regulatory mechanisms. They have also been the subject of spectacular advances in their structural understanding in recent years, as testified by the attribution of the Nobel prize in chemistry in 2006 to Roger Kornberg. The book focuses on two aspects of the transcription cycle that have been more intensively studied thanks to this increased scientific cooperation - the recognition of the promoter by the enzyme, and the achievement of consecutive translocation steps during elongation of the RNA product. Each of these two topics is introduced by an overview, and is then presented by worldwide experts in the field, taking the viewpoint of their speciality. The overview chapters focus on the mechanism-structure interface and the structure-machine interface while the individual chapters within each section concentrate more specifically on particular processes-kinetic analysis, single-molecule spectroscopy, and termination of transcription, amongst others. Specific attention has been paid to the newcomers in the field, with careful descriptions of new emerging techniques and the constitution of an atlas of three-dimensional pictures of the enzymes involved. For more than thirty years, the study of RNA polymerases has benefited from intense cooperation between the scientific partners involved in the various fields listed above. It is hoped that a collection of essays from outstanding scientists on this subject will catalyse the convergence of scientific efforts in this field, as well as contribute to better teaching at advanced levels in Universities.
There and Back Again: A Structural Atlas of RNAP | p. 1 |
From Promoter Recognition to Promoter Escape | |
Where it all Begins: An Overview of Promoter Recognition and Open Complex Formation | |
Gene Expression as a Driver of Life | p. 13 |
Escherichia coli RNA Polymerase | p. 14 |
Promoters and Core Promoter Elements | p. 17 |
Biochemistry: It Works with RNA Polymerase! | p. 19 |
Biochemistry of Promoter Regulation | p. 22 |
A Word about the Intracellular Environment | p. 26 |
Coupling Transcription to Changes in a Complex Environment | p. 27 |
A Global View of the RNA Polymerase Economy | p. 30 |
The Real World, Emergency Procedures and RNA Polymerase | p. 32 |
This is just the Beginning! | p. 33 |
References | p. 34 |
Opening the DNA at the Promoter; The Energetic Challenge | |
Introduction | p. 38 |
Structural Characterization | p. 42 |
Crystal Structure of the Holoenzyme | p. 42 |
Crystal Structure of the Holoenzyme with Fork-junction DNA | p. 43 |
Structural Model of the Open Complex | p. 45 |
Physical Characterization and Structure of the Intermediates | p. 47 |
Finding the Promoter. Induced fit and Indirect Sequence Recognition | p. 48 |
Formation of the Closed Complex | p. 49 |
On a Unique Structure of the Closed Complex | p. 49 |
Role of Upstream Contacts for the Stability of the Closed Complex and in Leading the Complex Towards Subsequent Isomerization | p. 50 |
The First Isomerization Step. The role of Sigma, Formation of Specific Interactions | p. 51 |
Upstream Contacts | p. 51 |
Nucleation of the Single Stranded Region and its Propagation | p. 52 |
Phasing of -10 and -35 Regions, the Role of the Spacer | p. 54 |
Probing Possible Sequential Linear Pathways by the use of Temperature | p. 54 |
Specific Protein Domains Destabilize the Intermediates in the Pathway | p. 57 |
Overstabilization is sometimes used as a Regulatory Mechanism | p. 59 |
Formation of Transcriptionally Active Open Complex and the Rate-limiting Step: Protein Conformational Changes or DNA Melting? | p. 60 |
A Rugged Energy Landscape | p. 61 |
Summary and Conclusions | p. 62 |
Acknowledgements | p. 63 |
References | p. 63 |
Intrinsic In vivo Modulators: Negative Supercoiling and the Constituents of the Bacterial Nucleoid | |
Introduction | p. 69 |
DNA Superhelicity - Structures and Implications | p. 69 |
Structure of the Bacterial Nucleoid | p. 72 |
Supercoiling Utilization | p. 75 |
Promoter Structure and DNA Supercoiling | p. 78 |
Role of RNA Polymerase Composition | p. 80 |
Exchange of Factors | p. 81 |
Auxiliary Subunits | p. 82 |
Role of ppGpp | p. 82 |
Model and Implications | p. 82 |
Causality | p. 85 |
Cooperation with Nucleoid Associated Proteins | p. 86 |
Conversion of Supercoil Energy into Genomic Transcript Patterns | p. 87 |
Conclusions | p. 88 |
References | p. 88 |
Transcription by RNA Polymerases: From Initiation to Elongation, Translocation and Strand Separation | |
Introduction | p. 96 |
Transition from the Initiation to the Elongation Phase | p. 98 |
T7 RNA Polymerase | p. 98 |
Multi-subunit RNA Polymerases | p. 103 |
Translocation and Strand Separation | p. 103 |
T7 RNA Polymerase | p. 103 |
Multi-subunit Cellular RNAPs | p. 108 |
Additional Similarities between Single and Multi-subunit Polymerases | p. 112 |
Acknowledgements | p. 113 |
References | p. 113 |
Single-molecule FRET Analysis of the Path from Transcription Initiation to Elongation | |
Introduction | p. 115 |
Methodology: FRET and ALEX Spectroscopy | p. 117 |
Transcription Mechanisms Addressed using Single-molecule FRET and ALEX | p. 124 |
Fate of Initiation Factor 70 in Elongation | p. 126 |
Mechanism of Initial Transcription | p. 133 |
Kinetic Analysis of Initial Transcription and Promoter Escape | p. 141 |
Comparison of FRET Approaches with Magnetic-trap Approaches | p. 142 |
Future Prospects | p. 145 |
Summary | p. 147 |
Acknowledgements | p. 148 |
References | p. 148 |
Real-time Detection of DNA Unwinding by Escherichia coli RNAP: From Transcription Initiation to Termination | |
Introduction | p. 157 |
Twist Deformations at the Promoter | p. 158 |
Magnetic Trapping and Supercoiling of a Single DNA Molecule | p. 159 |
General Features of the Magnetic Trap | p. 159 |
Calibrating the DNA Sensor | p. 161 |
Characterization of RPo at two Canonical Promoters | p. 166 |
Structural Characterization of RPo | p. 167 |
Kinetic Analysis of RPo | p. 168 |
Effect of Environmental Variables on Kinetics of RPo | p. 171 |
Promoter Escape by DNA Scrunching | p. 172 |
Characterization of DNA Scrunching during Abortive Initiation | p. 173 |
Characterization of DNA Scrunching during Promoter Escape | p. 176 |
Future Directions | p. 182 |
References | p. 183 |
Transcription Elongation and Termination | |
Interlude The Engine and the Brake | |
Introduction | p. 191 |
The Engine | p. 193 |
Mechano-chemical Coupling at the Catalytic Site | p. 194 |
Coupling between Translocation and Topology | p. 200 |
The Brake | p. 201 |
Conclusions | p. 202 |
References | p. 204 |
Substrate Loading, Nucleotide Addition, and Translocation by RNA Polymerase | |
Basic Mechanisms of Transcript Elongation by RNA Polymerase | p. 206 |
Active-site Features of an Elongation Complex | p. 207 |
The Nucleotide Addition Cycle | p. 207 |
Pyrophosphorolysis and Transcript Cleavage | p. 208 |
Regulation of Transcript Elongation by Pauses | p. 211 |
Structural Basis of NTP Loading and Nucleotide Addition | p. 212 |
Bridge-helix-centric Models of Nucleotide Addition and Translocation | p. 213 |
Central Role of the Trigger Loop in Nucleotide Addition and Pausing | p. 216 |
A Trigger-loop Centric Mechanism for Substrate Loading and Catalysis | p. 217 |
Models of Translocation: Power-stroke versus Brownian Ratchet | p. 219 |
Key Distinctions between Power-stroke and Brownian Ratchet Models | p. 220 |
Power-stroke Models | p. 221 |
Brownian Ratchet Models | p. 221 |
Technical Outlook in Detecting the Precise Translocation Register | p. 222 |
Kinetic Models of Nucleotide Addition | p. 223 |
Allosteric NTP Binding Model | p. 223 |
NTP-driven Translocation Model | p. 226 |
Two-pawl Ratchet Model | p. 226 |
Biophysical Models for Transcript Elongation | p. 227 |
Technological Advances in Studies of Transcript Elongation | p. 228 |
Concluding Remarks | p. 228 |
References | p. 229 |
Regulation of RNA Polymerase through its Active Center | |
Introduction | p. 236 |
Regulatory Checkpoints of the RNAP Active Center | p. 237 |
Versatility of the Active Center. How many Metals are Enough? | p. 237 |
Delivery of NTPs to the Active Center. How many Channels are Enough? | p. 238 |
Nucleotide Selection. How many Steps are Enough? | p. 241 |
Regulators that Target the RNAP Active Center | p. 244 |
Small-molecule Effectors of RNAP | p. 244 |
Regulation of RNAP by Proteins that Bind in the Secondary Channel | p. 250 |
Transcript Proofreading | p. 254 |
Transcriptional Proofreading through Pyrophosphorolysis | p. 255 |
Proofreading by Transcript Cleavage Factors | p. 256 |
Transcript-assisted Proofreading. A New Class of Ribozymes? | p. 257 |
Conclusions | p. 258 |
Acknowledgements | p. 259 |
References | p. 259 |
Kinetic Modeling of Transcription Elongation | |
Introduction | p. 263 |
Background | p. 265 |
Mechano-chemical Coupling of Transcription | p. 266 |
NTP Incorporation Cycle | p. 266 |
NTP Incorporation Pathway in a Simple Brownian Ratchet Model | p. 266 |
NTP Incorporation Pathways in more Elaborate Brownian Ratchet Models | p. 267 |
NTP Incorporation Pathway in a Power-stroke Model | p. 269 |
Elongation Kinetics | p. 270 |
Force-dependent Elongation Kinetics | p. 271 |
Sequence-dependent RNAP Kinetics | p. 274 |
Thermodynamic Analysis of the TEC | p. 274 |
Sequence-dependent NTP Incorporation Kinetics in Brownian Ratchet Models | p. 275 |
Model Predictions of Pause Locations, Kinetics and Mechanisms | p. 277 |
Acknowledgements | p. 278 |
References | p. 278 |
Mechanics of Transcription Termination | |
Introduction | p. 281 |
Structure/Function Overview of the Elongation Complex (EC) | p. 282 |
Mechanism of Intrinsic Termination | p. 283 |
The Pausing Phase | p. 285 |
The Termination Phase | p. 287 |
Mechanism of Rho Termination | p. 294 |
Summary | p. 295 |
Concluding Remarks | p. 296 |
References | p. 296 |
Conclusion Past, Present, and Future of Single-molecule Studies of Transcription | |
Introduction | p. 302 |
RNA Polymerase as a Molecular Machine: Past and Present | p. 303 |
Technical Developments in Optical Tweezers | p. 307 |
A Look into the Future | p. 309 |
References | p. 312 |
Subject Index | p. 315 |
Table of Contents provided by Ingram. All Rights Reserved. |
ISBN: 9780854041343
ISBN-10: 0854041346
Series: RSC Biomolecular Sciences
Published: 1st April 2009
Format: Hardcover
Language: English
Number of Pages: 331
Audience: Professional and Scholarly
Publisher: Royal Society Of Chemistry
Country of Publication: GB
Dimensions (cm): 23.4 x 15.6 x 2.54
Weight (kg): 0.67
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