Download or read book Single Molecule Studies of Bacterial Transcription Under Force and Torque Using Optical Tweezers written by Yara Xochitl Mejia and published by . This book was released on 2009 with total page 282 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Download or read book Handbook of Single Molecule Biophysics written by Peter Hinterdorfer and published by Springer Science & Business Media. This book was released on 2009-12-24 with total page 634 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Download or read book Single molecule Techniques written by Paul R. Selvin and published by CSHL Press. This book was released on 2008 with total page 511 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Download or read book Optical Tweezers written by Arne Gennerich and published by Springer Nature. This book was released on 2022-09-05 with total page 753 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Download or read book Biophysics of RNA Protein Interactions written by Chirlmin Joo and published by Springer Nature. This book was released on 2019-09-19 with total page 249 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Download or read book Optical Trapping And Manipulation Of Neutral Particles Using Lasers A Reprint Volume With Commentaries written by Arthur Ashkin and published by World Scientific. This book was released on 2006-12-29 with total page 941 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Download or read book Investigation of Bacterial Transcription Using Single Molecule Techniques written by SangYoon Chung and published by . This book was released on 2016 with total page 124 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Download or read book Micromanipulation by Light in Biology and Medicine written by Karl Otto Greulich and published by Birkhauser. This book was released on 1999 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Optical Tweezers written by Miles J. Padgett and published by CRC Press. This book was released on 2010-06-02 with total page 510 pages. Available in PDF, EPUB and Kindle. Book excerpt: The technical development of optical tweezers, along with their application in the biological and physical sciences, has progressed significantly since the demonstration of an optical trap for micron-sized particles based on a single, tightly focused laser beam was first reported more than twenty years ago. Bringing together many landmark papers on

Download or read book Bacterial Chromatin written by Remus T. Dame and published by Springer Nature. This book was released on with total page 654 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Introductory Biomechanics written by C. Ross Ethier and published by Cambridge University Press. This book was released on 2007-03-12 with total page 10 pages. Available in PDF, EPUB and Kindle. Book excerpt: Introductory Biomechanics is a new, integrated text written specifically for engineering students. It provides a broad overview of this important branch of the rapidly growing field of bioengineering. A wide selection of topics is presented, ranging from the mechanics of single cells to the dynamics of human movement. No prior biological knowledge is assumed and in each chapter, the relevant anatomy and physiology are first described. The biological system is then analyzed from a mechanical viewpoint by reducing it to its essential elements, using the laws of mechanics and then tying mechanical insights back to biological function. This integrated approach provides students with a deeper understanding of both the mechanics and the biology than from qualitative study alone. The text is supported by a wealth of illustrations, tables and examples, a large selection of suitable problems and hundreds of current references, making it an essential textbook for any biomechanics course.

Download or read book Advances In Atomic Physics An Overview written by Claude Cohen-tannoudji and published by World Scientific. This book was released on 2011-09-02 with total page 794 pages. Available in PDF, EPUB and Kindle. Book excerpt: “French Nobel Laureate Claude Cohen-Tannoudji is second to none in his understanding of the modern theory and application of atom-photon interactions. He is also known for his lucid and accessible writing style … Advances in Atomic Physics is an impressive and wonderful-to-read reference text … Certainly researchers in the fields of atom-photon interactions and atom traps will want it as a reference on their bookshelves … A selection of chapters may be of benefit to students: the early chapters for those entering the field, the later chapters for those already doing atom-laser PhD thesis work.”Physics TodayThis book presents a comprehensive overview of the spectacular advances seen in atomic physics during the last 50 years. The authors explain how such progress was possible by highlighting connections between developments that occurred at different times. They discuss the new perspectives and the new research fields that look promising. The emphasis is placed, not on detailed calculations, but rather on physical ideas. Combining both theoretical and experimental considerations, the book will be of interest to a wide range of students, teachers and researchers in quantum and atomic physics.

Download or read book RNA Polymerases as Molecular Motors written by Robert Landick and published by Royal Society of Chemistry. This book was released on 2021-11-23 with total page 295 pages. Available in PDF, EPUB and Kindle. Book excerpt: To thrive, every living cell must continuously gauge and respond to changes in its environment. These changes are ultimately implemented by modulating gene expression, a process that relies on transcription by Nature’s most multivalent molecular machine, the RNA polymerase. This book covers progress made over the past decade understanding how this machine functions to compute the cellular state, from the atomistic structural level responsible for chemistry to the integrative level at which RNA polymerase interacts with the other key molecular machineries of the cell.

Download or read book Single Molecule and Synthetic Biology Studies of Transcription written by Bradley Michael Zamft and published by . This book was released on 2011 with total page 326 pages. Available in PDF, EPUB and Kindle. Book excerpt: The horizons of biology are ever expanding, from the discernment of the detailed mechanisms of enzyme function, to the manipulation of the physiological processes of whole organisms and ecosystems. Single molecule studies allow for the characterization of the individual processes that comprise an enzyme's mechanochemical cycle. Through standardization and generalization of biological techniques, components, and knowledge, synthetic biology seeks to expand the scale of biological experiments and to usher in an age of biology as a true engineering science, in which those studying different hierarchical levels of sophistication need not start from the fundamental biochemical principles underlying all biological experiments. Here we report our findings on the processes governing transcription and its role in gene expression through the use of both single molecule and synthetic biology methods. We have established a promoter-free, factor-free method of initiation of transcription by the mitochondrial RNA polymerase in Saccharomyces cerevisiae, Rpo41 through the use of synthetic oligonucleotides to imitate the hybridization geometry of Rpo41 during active transcription. Using this system, we have established that a sub-micromolar NTP concentration is appropriate for non-saturating transcriptional runoff assays. We have optimized the transcription buffer and found that 10 mM MgCl2, 40 mM KCl, and 10 mM DTT are sufficient for robust transcription. Stability studies show that Rpo41 loses approximately 30% of its activity during each freeze-thaw cycle, and that the pre-formed elongation complex loses transcriptional activity with a half-life of 7.4±1.5 hr. Through the use of optical trapping techniques, we have established a method to monitor the transcription of individual Rpo41 molecules in real time. This has allowed us to measure the kinetic rates of nucleotide incorporation by the enzyme: Km = 22±13 μM-1 and vmax = 25±2.5 bp/s. Both of these rates are more similar to those of the main nuclear RNA polymerase in the same organism, RNA Polymerase II (Pol II) than to that of the T7 RNA polymerase, despite the fact that Rpo41 is a single-subunit RNA polymerase with homology to those of the T-odd bacteriophage and no discernable homology to Pol II. Furthermore, like Pol II and the E. coli RNA polymerase, transcription by Rpo41 consists of periods of processive transcription interspersed with periods of pausing. We have also observed retrograde motion of Rpo41 during pauses, termed backtracking, a process that has not been reported in phage-like RNA polymerases. We have performed single molecule assays of transcription by both Pol II and Rpo41 on templates of differing base pair composition and found that, in general, the characteristics of pausing are attenuated in templates of higher GC content. Specifically, the frequency of pausing is decreased in GC-rich templates, as is the average pause duration. The distribution of pause durations is correspondingly shifted to shorter pauses on GC-rich templates. We discuss two mechanisms by which template composition may affect pausing: (1) movement of the backtracked transcription bubble is affected by differences in the base stacking energies from the disrupted/created DNA/DNA and RNA/DNA base pairs at the ends of the bubble, and (2) secondary structure of the nascent RNA upstream of the backtracked transcription bubble imposes an energetic barrier to its backward movement. We give in silico evidence that it is the latter mechanism. Incorporation of this secondary structure energy barrier (an "energy penalty") into a model of transcriptional pausing by backtracking allows for statistical fits of the mean pause densities, mean pause durations, and the distribution of pause durations for each enzyme on each template. Furthermore, incorporation of the energy penalty allows for fitting of the pause characteristics for a given enzyme using a single, enzyme specific hopping rate, k0, that is independent of template, and a single, template dependent energy penalty term, [Delta]GRNA, which is enzyme independent. For Rpo41, we find that k0, the hopping rate of the backtracked enzyme along DNA without RNA secondary structure, is 5.4±1.8 s-1, while it is 2.9±0.3 s-1 for Pol II. Furthermore, the average energy penalty due to the nascent RNA, [Delta]GRNA, on the AT-rich template used in this study is 0.7±0.1 kT, while it is 0.8±0.1 kT for random DNA and 1.0±0.1 kT for GC-rich DNA. In order to confirm that it is the secondary structure of the RNA that is the cause of the energy penalty, we performed the same single-molecule transcription assays in the presence of RNase A, an enzyme that digests unprotected RNA in both single-stranded and double-stranded form. The pausing characteristics of all traces on all templates in the presence of RNase A are statistically indistinguishable from those on AT-rich DNA without RNase, indicating that the RNase digested enough of the nascent RNA to disrupt any secondary structure. Protection of the 5' region of the nascent RNA by steric interactions between the polymerase and the RNase prevented full degradation of the RNA, and thus allowed for some backtracking. This strongly supports the new model, presented here, of modulation of transcriptional pausing by secondary structure of the nascent RNA. In contrast to the detailed and isolated nature of single-molecule transcription, we also performed a synthetic biology project involving Rpo41. The intent of this project was to investigate the plausibility of the creation of a transcriptionally independent mitochondrion, and by extension a minimal cell, by movement of the mitochondrial transcriptional machinery from the nuclear to the mitochondrial genome. Thus we performed in vivo mitochondrial transformation of yeast cells with a synthetic construct containing the gene encoding for Rpo41. We report that we have successfully integrated said synthetic gene into the mitochondrial genome, and have seen its expression to the transcriptional level. Furthermore, we are fairly confident that the full, intact mRNA of the synthetic gene is being created within the mitochondrial matrix. We have not been able to detect expression of the protein product of the integrated synthetic construct, nor have we been able to isolate a strain that exhibits its expression in the absence of the wild-type, nuclear copy. Because the length of Rpo41 is longer than any other protein synthesized within the mitochondrial organelle, we have begun experiments to determine the maximal polypeptide length able to be translated by the mitochondrial ribosome and associated cofactors.

Download or read book Structure and Dynamics of Confined Polymers written by John J. Kasianowicz and published by Springer Science & Business Media. This book was released on 2002-07-31 with total page 46 pages. Available in PDF, EPUB and Kindle. Book excerpt: Polymers are essential to biology because they can have enough stable degrees of freedom to store the molecular code of heredity and to express the sequences needed to manufacture new molecules. Through these they perform or control virtually every function in life. Although some biopolymers are created and spend their entire career in the relatively large free space inside cells or organelles, many biopolymers must migrate through a narrow passageway to get to their targeted destination. This suggests the questions: How does confining a polymer affect its behavior and function? What does that tell us about the interactions between the monomers that comprise the polymer and the molecules that confine it? Can we design and build devices that mimic the functions of these nanoscale systems? The NATO Advanced Research Workshop brought together for four days in Bikal, Hungary over forty experts in experimental and theoretical biophysics, molecular biology, biophysical chemistry, and biochemistry interested in these questions. Their papers collected in this book provide insight on biological processes involving confinement and form a basis for new biotechnological applications using polymers. In his paper Edmund DiMarzio asks: What is so special about polymers? Why are polymers so prevalent in living things? The chemist says the reason is that a protein made of N amino acids can have any of 20 different kinds at each position along the chain, resulting in 20 N different polymers, and that the complexity of life lies in this variety.

Download or read book Visualizing Chemistry written by National Research Council and published by National Academies Press. This book was released on 2006-06-01 with total page 222 pages. Available in PDF, EPUB and Kindle. Book excerpt: Scientists and engineers have long relied on the power of imaging techniques to help see objects invisible to the naked eye, and thus, to advance scientific knowledge. These experts are constantly pushing the limits of technology in pursuit of chemical imagingâ€"the ability to visualize molecular structures and chemical composition in time and space as actual events unfoldâ€"from the smallest dimension of a biological system to the widest expanse of a distant galaxy. Chemical imaging has a variety of applications for almost every facet of our daily lives, ranging from medical diagnosis and treatment to the study and design of material properties in new products. In addition to highlighting advances in chemical imaging that could have the greatest impact on critical problems in science and technology, Visualizing Chemistry reviews the current state of chemical imaging technology, identifies promising future developments and their applications, and suggests a research and educational agenda to enable breakthrough improvements.

Download or read book Molecular Motors written by Christophe Lavelle and published by Humana. This book was released on 2019-07-18 with total page 452 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.