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: 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: Yazan Khalaf Alhadid
Publisher:
ISBN:
Category :
Languages : en
Pages : 146
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Book Description
Transcription of genomic DNA of all organisms is carried out by members of the multi-subunit RNA polymerase family. Regulation of RNA polymerase localization and activity underlies cellular homeostasis, division, and response to environmental cues. The catalytic mechanism, overall architecture, and many sequence and structural features of bacterial RNA polymerase are conserved in its Archaeal and Eukaryotic counterparts. The human RNA polymerase II (Pol II) is responsible for transcription of all protein-coding and many non-coding genes. The majority of current knowledge on RNA polymerases and their mechanism at different steps in transcription derives from extensive work done using classical biochemical, genetic and structural biology methods. However, the use of single-molecule approaches addressed crucial questions on the function and mechanism of RNA polymerases during transcription, which were not possible to answer with ensemble-based approaches due to averaging effects. A useful fluorescence-based single-molecule technique to measure distances on the molecular scale and monitor dynamics is F rster resonance energy transfer (FRET). Here, I report on the development of diffusion-based single-molecule FRET (smFRET) methods to investigate different steps in transcription by the in vitro reconstituted human Pol II system. Using an assay that monitors the FRET changes between fluorescent dyes in the unwound region of promoter DNA (transcription bubble), I demonstrated the effect of certain components of the reconstituted system on the relative size of the transcription bubble. I also detail the optimizations done to enhance the affinity of single-stranded DNA (ssDNA) FRET probes to complementary target sequences. These ssDNA FRET probes were used to investigate the effect of certain components of the reconstituted system on Pol II activity by measuring the relative levels of RNA product. In addition to studies on the Pol II system, I report on the effect of the 5'-group of nascent RNA on the stability of the Escherichia coli RNA polymerase (RNAP) transcription bubble. I show how the presence of a 5'-monophosphate appears to destabilize the open bubble while a 5'-hydroxyl has no effect. Finally, I describe the work done on a project I took part in that identified a previously uncharacterized RNAP paused complex in initiation. We demonstrate that RNAP complexes undergoing initial transcription can enter the inactive paused state by backtracking. I also demonstrate how the presence of a 5'-triphosphate rapidly enhances entrance of RNAP complexes undergoing initial transcription into an inactive paused complex.
Author: Ming Li
Publisher:
ISBN:
Category :
Languages : en
Pages : 146
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Book Description
Biological systems create designs that respond to the need to perform specific functions. In particular, protein-DNA complexes form unique structures to maintain the stability of genetic information and yet the dynamics for necessary cellular processes. Motor proteins translocate along, and rotate around, DNA molecules to separate DNA strands, carry out polymerization reactions, resolve topological issues, repair DNA damage, and modify DNA-binding proteins. By investigating one molecular complex at a time, single molecule techniques provide controlled and quantitative approaches to measure and manipulate the protein-DNA interactions as well as visualize the function of motor proteins in real time. These techniques have now made it possible to address many problems that are difficult or impossible to study with traditional assays In this dissertation, we first introduce DNA unzipping as a powerful tool to study protein-DNA interactions at the single-molecule level. In particular, we detail protocols for preparing an unzipping template, constructing and calibrating the instrument, and acquiring, processing, and analyzing unzipping data. We also summarize major results from utilizing this technique in the studies of nucleosome structures and dynamics. After that, we use DNA unzipping to systematically investigate the interplay between nucleosome remodeling and the binding of transcription factors. The results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes. In the last chapter, we elaborate the single molecule unzipping tracker technique and its application in understanding the function of the bacterial transcription coupled repair factor Mfd. The results provide important insights into the role of Mfd beyond the scope of transcription coupled repair and significantly contribute to the understanding of Mfd function in the larger context of transcription.
Author:
Publisher:
ISBN:
Category : DNA polymerases
Languages : en
Pages : 145
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Book Description
Transcription involves many reaction steps and intermediates. Many phenomena in transcription kinetics are covered by ensemble average. Single-molecule DNA nanomanipulation techniques uncover these transcription kinetic events via determination of a transcription bubble in real time. In this dissertation, we focus on the transcription kinetics of bacterial RNAP. The study of transcription kinetics in this thesis can be divided into 4 main subjects: 1) The study of abortive initiation mechanism: Through a single-molecule DNA nanomanipulation technique, we tested the three models proposed for the mechanism of abortive initiation - inchworming, scrunching and transient excursion - on T5 N25 promoter. Of the three models, only the scrunching model involves a change in the size of the transcription bubble during abortive initiation, which was observed by single-molecule DNA nanomanipulation technique. 2) The study of the kinetics of elongation and termination: By introducing varying transcribed region lengths into DNA templates, the kinetics of elongation and terminator rewinding were studied. The resulting kinetics, determined by the single-molecule nanomanipulation technique, was analyzed by different regression methods. In the normal regression, the elongation velocity is 10 b/s and terminator rewinding takes 3.4 s on a tR2 terminator. Contrarily, through Poisson regression, the elongation velocity ranges from 6.4 b/s to 12.5 b/s and terminator rewinding takes 11.9 s on a tR2 terminator. 3) The study of a promoter sequence's effect: The effect from sequence of the T5 N25 promoter and T5 N25 antiDSR promoter in transcription was evaluated. The transcription bubble sizes of open complex and initial transcribing complex using the T5 N25 antiDSR promoters are larger than the ones from the T5 N25 promoter. The difference in the two promoter sequences does not have an effect on the transcription bubble size of an elongation complex. And elongation and terminator rewinding kinetics are not affected. On the other hand, abortive initiation and promoter escape are affected by the difference in the promoter sequence. 4) The study of the effect of transcription factor-GreB: The effect of transcription factor-GreB on abortive initiation was evaluated by the single-molecule DNA nanomanipulation technique. GreB does not affect the transcription bubble size during abortive initiation, but does reduce the lifetime of initial transcribing complex.
Author: Matthew Herbert Larson
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
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: 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.
Author: Soohong Kim
Publisher:
ISBN:
Category :
Languages : en
Pages : 112
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Book Description
Author: Hua Yu
Publisher:
ISBN:
Category :
Languages : en
Pages : 306
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Book Description
Author: Irina Artsimovitch
Publisher: Humana
ISBN: 9781493954674
Category : Science
Languages : en
Pages : 0
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Book Description
This volume is designed to be a resource of proven techniques and approaches for probing the activities of bacterial, eukaryotic, and archaeal RNA polymerases. This book features a collection of in vitro and in vivo technologies that will permit researchers to purify and probe the position and stability of RNA polymerase complexes at different points of the transcription cycle, analyze the various translocations and intermolecular movements associated with catalysis, define recruitment strategies, probe the roles of transcription factors in each stage of the cycle, highlight conserved and disparate fidelity mechanisms, analyze the resultant transcripts, and study coordination of the nascent mRNA synthesis by the RNA polymerase and mRNA translation by the ribosome. Written in the highly successful Methods of 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 key tips on troubles troubleshooting and avoiding known pitfalls. Practical and timely, Bacterial Transcriptional Controls: Methods and Protocols highlights the breadth and depth of techniques that are likely to continue shaping the transcription community in the future.
