Author: Yara Xochitl Mejia
Publisher:
ISBN:
Category :
Languages : en
Pages : 282
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Book Description
The present dissertation uses Optical Tweezers to examine the inner workings of one of the most relevant molecular motors in the cell: RNA polymerase. E. coli RNA polymerase has been studied for almost half a century, but fundamental questions about its translocation mechanism along DNA, as well as its pausing and stalling behavior still remain. Due to RNAP's heterogeneous behavior, a single molecule approach presents unique advantages. As part of this work, single molecule transcription experiments were done at temperatures between 7°C and 45°C. Within this temperature range, the pause-free velocity of RNAP increases with temperature with an activation energy of 9.7 ± 0.7 kcal/mole. Moreover, temperature affects pause entry and the stalling force, but not pause duration. This dissertation also presents the first single molecule study of Trigger Loop (TL) mutants, a domain thought to have a crucial role in enzyme catalysis. Our results identify TL folding as a rate-determining step in elongation and correlate TL helix propensity with pause-free velocity. Based on the inverse relation between pause-free velocity and pause entry for the mutant and wildtype polymerases and for transcription with nucleotide analogs, a quantitative kinetic model was constructed. An analysis of pause durations indicated that the TL has no role in pause recovery. Furthermore, a novel single molecule assay was developed to study RNAP's rotation velocity during elongation and in response to torsional load. Here, the DNA is stretched between two beads, and a "rotor bead" of different sizes is attached to the rotating polymerase. The pause-free angular speed is seen to decrease for increasing rotational loads corresponding to a constant torque value of 7 pNnm. Further analysis demonstrates that RNAP acts as a Brownian Ratchet and exerts an average energy per step of 1 KBT. Rotational load does not, however, have an effect on pause entry or duration. Finally, I describe a novel technique for modifying the twist of DNA. This Hybrid Tweezers technique was used for the formation of DNA plectonemes and braids, as well as for studying transcription under Torque. Together, these experiments have constructed a clearer picture of the kinetics, energetics and mechanisms of RNAP.
Author: Peter Hinterdorfer
Publisher: Springer Science & Business Media
ISBN: 0387764976
Category : Science
Languages : en
Pages : 634
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Book Description
This handbook describes experimental techniques to monitor and manipulate individual biomolecules, including fluorescence detection, atomic force microscopy, and optical and magnetic trapping. It includes single-molecule studies of physical properties of biomolecules such as folding, polymer physics of protein and DNA, enzymology and biochemistry, single molecules in the membrane, and single-molecule techniques in living cells.
Author: Paul R. Selvin
Publisher: CSHL Press
ISBN: 087969775X
Category : Science
Languages : en
Pages : 511
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Book Description
Geared towards research scientists in structural and molecular biology, biochemistry, and biophysics, this manual will be useful to all who are interested in observing, manipulating and elucidating the molecular mechanisms and discrete properties of macromolecules.
Author: Arne Gennerich
Publisher: Springer Nature
ISBN: 1071622293
Category : Science
Languages : en
Pages : 753
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Book Description
This detailed volume explores a wide variety of techniques involving optical tweezers, a technology that has become increasingly more accessible to a broad range of researchers. Beginning with recent technical advances, the book continues by covering the application of optical tweezers to study DNA-protein interactions and DNA motors, protocols to perform protein (un)folding experiments, the application of optical tweezers to study actin- and microtubule-associated motor proteins, and well as protocols for investigating the function and mechanical properties of microtubules and intermediate filaments, and more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Optical Tweezers: Methods and Protocols, Second Edition serves as an ideal resource for expanding the accessibility and use of optical traps by scientists of diverse disciplines.
Author: SangYoon Chung
Publisher:
ISBN:
Category :
Languages : en
Pages : 124
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Book Description
The numerous complex molecular processes occurring inside living cells are primarily carried out by proteins and other biopolymers, such as ribonucleic acids (RNA). The identity and quantity of the different proteins and RNA determine the cell's phenotype and changes in response to the environment. Therefore, the internal composition of the cell in terms of the type and concentration of proteins and RNA is tightly regulated. Gene expression is the process of using the DNA sequence information to produce these biopolymers. Transcription, the initial step in gene expression, where one strand of DNA is used as template by the enzyme RNA polymerase (RNAP) for synthesizing a complementary RNA or transcript. Since cell phenotype is mostly determined by transcription, a complex regulatory mechanism exists involving a large number of factors to control the level of transcription of a gene. Although most studies are focused on multiple cycles of either transcription or association of DNA and RNA Polymerase (RNAP) to make RNAP-Promoter open complex (RPO), single round transcription studies are crucial in elucidating the mechanism of sophisticated RNAP-DNA interactions and its kinetics in transcription. In this context, we have developed a novel in vitro quenching based single round transcription assay using single molecule detection. Using this, we could successfully dissect initiation kinetics starting from different initial transcribing stages and found that transcription initiation doesn't follow a sequential model (as commonly believed). Instead, we identified a previously uncharacterized state that is unique to initial transcribing complexes and associated with the backtracked RNAP-DNA complex. Also, we have investigated the size/concentration effects of various osmolytes and macromolecular crowding agents, which mimic the crowded cellular environment, on actively-transcribing RNAP and found enhancement in transcription kinetics by larger crowding agents at the same viscosity.
Author: Arthur Ashkin
Publisher: World Scientific
ISBN: 9814494348
Category : Science
Languages : en
Pages : 941
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Book Description
This important volume contains selected papers and extensive commentaries on laser trapping and manipulation of neutral particles using radiation pressure forces. Such techniques apply to a variety of small particles, such as atoms, molecules, macroscopic dielectric particles, living cells, and organelles within cells. These optical methods have had a revolutionary impact on the fields of atomic and molecular physics, biophysics, and many aspects of nanotechnology.In atomic physics, the trapping and cooling of atoms down to nanokelvins and even picokelvin temperatures are possible. These are the lowest temperatures in the universe. This made possible the first demonstration of Bose-Einstein condensation of atomic and molecular vapors. Some of the applications are high precision atomic clocks, gyroscopes, the measurement of gravity, cryptology, atomic computers, cavity quantum electrodynamics and coherent atom lasers.A major application in biophysics is the study of the mechanical properties of the many types of motor molecules, mechanoenzymes, and other macromolecules responsible for the motion of organelles within cells and the locomotion of entire cells. Unique in vitro and in vivo assays study the driving forces, stepping motion, kinetics, and efficiency of these motors as they move along the cell's cytoskeleton. Positional and temporal resolutions have been achieved, making possible the study of RNA and DNA polymerases, as they undergo their various copying, backtracking, and error correcting functions on a single base pair basis.Many applications in nanotechnology involve particle and cell sorting, particle rotation, microfabrication of simple machines, microfluidics, and other micrometer devices. The number of applications continues to grow at a rapid rate.The author is the discoverer of optical trapping and optical tweezers. With his colleagues, he first demonstrated optical levitation, the trapping of atoms, and tweezer trapping and manipulation of living cells and biological particles.This is the only review volume covering the many fields of optical trapping and manipulation. The intention is to provide a selective guide to the literature and to teach how optical traps really work.
Author: Christophe Lavelle
Publisher: Humana
ISBN: 9781493993246
Category : Science
Languages : en
Pages : 452
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Book Description
This volume expands on the previous edition with a more extensive look at molecular motors and their roles in muscle contractions, vesicle transport, flagellar beating, chromosome segregation, and DNA replication and repair. The chapters in this book are divided into three parts: Part One looks at membrane motors, such as the bacterial flagellar rotary motor; Part Two discusses cytoskeletal motors, such as kinesin and myosin; and Part Three talks about nucleic acid motors, such as DNA polymerases, helicases, and nucleosome remodelers. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Molecular Motors: Methods and Protocols, Second Edition is a valuable resource for (bio)physicists and molecular/cellular biologists whose research delves into the mechanisms at work in cells and the motors which power them.
Author: Chirlmin Joo
Publisher: Springer Nature
ISBN: 1493997262
Category : Science
Languages : en
Pages : 249
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Book Description
RNA molecules play key roles in all aspects of cellular life, but to do so efficiently, they must work in synergism with proteins. This book addresses how proteins and RNA interact to carry out biological functions such as protein synthesis, regulation of gene expression, genome defense, liquid phase separation and more. The topics addressed in this volume will appeal to researchers in biophysics, biochemistry and structural biology. The book is a useful resource for anybody interested in elucidating the molecular mechanisms and discrete properties of RNA-protein complexes. Included are reviews of key systems such as microRNA and CRISPR/Cas that exemplify how RNA and proteins work together to perform their biological function. Also covered are techniques ranging from single molecule fluorescence and force spectroscopy to crystallography, cryo-EM microscopy, and kinetic modeling.
Author: Remus T. Dame
Publisher: Springer Nature
ISBN: 1071639307
Category :
Languages : en
Pages : 654
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Book Description
Author: Chris R. Calladine
Publisher: Elsevier
ISBN: 0080474667
Category : Science
Languages : en
Pages : 349
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Book Description
The functional properties of any molecule are directly related to, and affected by, its structure. This is especially true for DNA, the molecular that carries the code for all life on earth. The third edition of Understanding DNA has been entirely revised and updated, and expanded to cover new advances in our understanding. It explains, step by step, how DNA forms specific structures, the nature of these structures and how they fundamentally affect the biological processes of transcription and replication. Written in a clear, concise and lively fashion, Understanding DNA is essential reading for all molecular biology, biochemistry and genetics students, to newcomers to the field from other areas such as chemistry or physics, and even for seasoned researchers, who really want to understand DNA. - Describes the basic units of DNA and how these form the double helix, and the various types of DNA double helix - Outlines the methods used to study DNA structure - Contains over 130 illustrations, some in full color, as well as exercises and further readings to stimulate student comprehension
