Single-molecule Studies on Transcriptional Elongation in Prokaryotes and Eukaryotes PDF Download
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Author: Jing Zhou
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Languages : en
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Transcription, the process of copying genetic information stored in DNA into RNA, is fundamental to life. It is carried out by an extraordinary nano-machine called RNA polymerase (RNAP). Transcriptional elongation, during which RNAP moves along the DNA, adding one nucleotide at a time to the RNA transcript, is highly dynamic and regulated. The motion of RNAP is discontinuous and interrupted by pauses that play an essential role in gene regulation. Fundamental questions regarding the mechanisms of elongation and its modulation by transcription factors, however, remain unclear. In this dissertation, I focus on using high-resolution, optical trapping techniques to study the mechanisms of transcriptional elongation by both prokaryotic and eukaryotic RNA polymerases at the single-molecule level. First, I describe the studies on how the motion of single E.coli RNAP molecules is modulated by two universally conserved, essential transcription factors (NusA and NusG). From individual transcriptional elongation records, the rates of entering pause states, the pause state lifetimes, and the pause-free elongation speeds can all be extracted. By studying the effects of NusA (and NusG) on these kinetic rates as a function of the applied load, we were able to develop a quantitative kinetic scheme for elongation and pausing. This model not only explains the functions of NusA/NusG, but also provides insight into the mechanism of transcriptional pausing, which had previously been controversial. Second, a novel optical-trapping assay capable of directly probing elongation by individual eukaryotic RNA polymerase II (RNAPII) molecules will be described. We find that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, not only affects each of the three main phases of elongation, namely: substrate binding, translocation, and catalysis; but also plays a critical role in controlling the fidelity of transcription. Our data also support a Brownian ratchet model for elongation which incorporates a secondary nucleotide binding site.
Author: Jing Zhou
Publisher:
ISBN:
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Languages : en
Pages :
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Book Description
Transcription, the process of copying genetic information stored in DNA into RNA, is fundamental to life. It is carried out by an extraordinary nano-machine called RNA polymerase (RNAP). Transcriptional elongation, during which RNAP moves along the DNA, adding one nucleotide at a time to the RNA transcript, is highly dynamic and regulated. The motion of RNAP is discontinuous and interrupted by pauses that play an essential role in gene regulation. Fundamental questions regarding the mechanisms of elongation and its modulation by transcription factors, however, remain unclear. In this dissertation, I focus on using high-resolution, optical trapping techniques to study the mechanisms of transcriptional elongation by both prokaryotic and eukaryotic RNA polymerases at the single-molecule level. First, I describe the studies on how the motion of single E.coli RNAP molecules is modulated by two universally conserved, essential transcription factors (NusA and NusG). From individual transcriptional elongation records, the rates of entering pause states, the pause state lifetimes, and the pause-free elongation speeds can all be extracted. By studying the effects of NusA (and NusG) on these kinetic rates as a function of the applied load, we were able to develop a quantitative kinetic scheme for elongation and pausing. This model not only explains the functions of NusA/NusG, but also provides insight into the mechanism of transcriptional pausing, which had previously been controversial. Second, a novel optical-trapping assay capable of directly probing elongation by individual eukaryotic RNA polymerase II (RNAPII) molecules will be described. We find that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, not only affects each of the three main phases of elongation, namely: substrate binding, translocation, and catalysis; but also plays a critical role in controlling the fidelity of transcription. Our data also support a Brownian ratchet model for elongation which incorporates a secondary nucleotide binding site.
Author: Matthew Herbert Larson
Publisher:
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Languages : en
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Transcription by RNAP is highly regulated in both prokaryotic and eukaryotic cells, and the ability of the cell to differentiate and respond to its environment is largely due to this regulation. During elongation, for example, RNAP is known to momentarily halt in response to certain cellular signals, and this pause state has been implicated in the regulation of gene expression in both prokaryotic and eukaryotic organisms. In addition, once RNAP reaches the end of a gene, it must reliably terminate and release the newly-transcribed RNA, providing another potential point of regulation within different cell types. Both of these steps are crucial to ensure proper gene expression. In this dissertation, I focus on transcription elongation by both prokaryotic and eukaryotic RNA polymerases, as well as their regulation through pausing and termination. To probe the role of RNA hairpins in transcriptional pausing, a novel single-molecule "RNA-pulling" assay was used to block the formation of secondary structure in the nascent transcript. Force along the RNA did not significantly affect transcription elongation rates, pause frequencies, or pause lifetimes, indicating that short "ubiquitous" pauses are not a consequence of RNA hairpins. Force-based single-molecule techniques were also used to study the mechanism and energetics of transcription termination in bacteria. The data suggest two separate mechanisms for termination: one that involves hypertranslocation of RNAP along the DNA, and one that involves shearing of the RNA:DNA hybrid within the enzyme. In addition, a quantitative energetic model is presented that successfully predicts the termination efficiency of both wild-type and mutant terminators. Finally, the implementation of a novel optical-trapping assay capable of directly observing transcription by eukaryotic RNA polymerase II (RNAPII) molecules is described. This approach was used to probe the RNAPII nucleotide-addition cycle, as well as the role of the trigger loop (a conserved subdomain) in elongation. The results are consistent with a Brownian ratchet model of elongation which incorporates a secondary NTP binding site, and the trigger loop was found to modulate translocation, NTP binding, and catalysis, as well as substrate selection and mismatch recognition by RNAPII.
Author: Furqan Muhammad Fazal
Publisher:
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Category :
Languages : en
Pages :
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Author: Julianne Zedalis
Publisher:
ISBN: 9781947172401
Category : Biology
Languages : en
Pages : 1923
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Biology for AP® courses covers the scope and sequence requirements of a typical two-semester Advanced Placement® biology course. The text provides comprehensive coverage of foundational research and core biology concepts through an evolutionary lens. Biology for AP® Courses was designed to meet and exceed the requirements of the College Board’s AP® Biology framework while allowing significant flexibility for instructors. Each section of the book includes an introduction based on the AP® curriculum and includes rich features that engage students in scientific practice and AP® test preparation; it also highlights careers and research opportunities in biological sciences.
Author: Kevin Oliver Kramm
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Languages : en
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Author:
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ISBN: 9780815332183
Category : Cells
Languages : en
Pages : 0
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Author: Bradley Michael Zamft
Publisher:
ISBN:
Category :
Languages : en
Pages : 326
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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.
Author: Yara Xochitl Mejia
Publisher:
ISBN:
Category :
Languages : en
Pages : 282
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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: Ron Milo
Publisher: Garland Science
ISBN: 1317230698
Category : Science
Languages : en
Pages : 400
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Book Description
A Top 25 CHOICE 2016 Title, and recipient of the CHOICE Outstanding Academic Title (OAT) Award. How much energy is released in ATP hydrolysis? How many mRNAs are in a cell? How genetically similar are two random people? What is faster, transcription or translation?Cell Biology by the Numbers explores these questions and dozens of others provid
Author: Imre Gaspar
Publisher: Humana Press
ISBN: 9781493972128
Category : Medical
Languages : en
Pages : 492
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Book Description
This volume introduces different concepts and methods of detecting RNA in biological material in a variety of model systems. The chapters in this book discuss methods that will answer numerous biological questions that arise in the study of RNAs. Some of the topics covered in this book are single mRNA molecule detection in embryos and neurons; detection of mRNA and associated molecules by ISH-IEM on frozen sections; optimizing molecular beacons for intracellular analysis of RNA; imaging translation dynamics of single mRNA molecules in live cells; preparation of high-throughput sequencing libraries; and capturing RNA binding proteins in embryos and in cell-culture. 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, RNA Detection: Methods and Protocols is a valuable resource for novel and experiences scientist in the expanding field of RNAs.
