Role of Nusg, Nusa, and Nascent Rna Structures in Regulating Transcription Elongation and Termination PDF Download
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Author: Expery O. Omollo
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
RNA polymerase (RNAP) often pauses during transcription to regulate gene expressionacross all life forms. This pausing behavior is regulated by specific DNA sequences, RNA structures, and transcription factors. This work investigates the bacterial RNAP pausing mechanism, particularly at hairpin-stabilized pauses, and how NusG and NusA proteins modulate RNAP pausing. Additionally, this study also examines the mechanism of intrinsic transcription termination in bacteria and how it's modulated by NusG and NusA. Both hairpin-stabilized pausing and intrinsic termination begin when RNAP pauses momentarily to enable the nascent RNA to fold inside the exit channel. However, these processes differ in the proximity and base composition of the hairpin structure to the RNA 3' end. Pause hairpins are positioned 11 or 12 bases away from the RNA 3' end, with non-specific bases in between whereas terminator hairpins form 7 or 8 bases away, with a U-tract in the RNA of the RNA-DNA hybrid. Using biochemical approaches, and cryo-electron microscopy, this study demonstrates that the RNA exit channel widens to accommodate RNA hairpin formation. This expansion causes a mobile portion of RNAP to rotate relative to its static portion, adopting an inactive "swiveled state" that halts nucleotide incorporation. Although certain transcription factors can rescue swiveled and paused RNAP, this work reveals that NusA stabilizes RNAP in its swiveled and paused state. In contrast, NusG shows species-specific effects: in Mycobacterium tuberculosis, it stabilizes the swiveled and paused state, whereas in Escherichia coli, it reverses the swivel, promoting RNAP elongation. For RNAP paused at intrinsic terminators, this study shows that RNAP must rewind the transcription bubble to allow the terminator hairpin to fully form. A complete terminator hairpin triggers the release of RNA and then DNA. These findings define a fundamental structural mechanism of bacterial RNAP during hairpin-stabilized pausing and intrinsic termination, providing a broader understanding of transcription regulation.
Author: Expery O. Omollo
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
RNA polymerase (RNAP) often pauses during transcription to regulate gene expressionacross all life forms. This pausing behavior is regulated by specific DNA sequences, RNA structures, and transcription factors. This work investigates the bacterial RNAP pausing mechanism, particularly at hairpin-stabilized pauses, and how NusG and NusA proteins modulate RNAP pausing. Additionally, this study also examines the mechanism of intrinsic transcription termination in bacteria and how it's modulated by NusG and NusA. Both hairpin-stabilized pausing and intrinsic termination begin when RNAP pauses momentarily to enable the nascent RNA to fold inside the exit channel. However, these processes differ in the proximity and base composition of the hairpin structure to the RNA 3' end. Pause hairpins are positioned 11 or 12 bases away from the RNA 3' end, with non-specific bases in between whereas terminator hairpins form 7 or 8 bases away, with a U-tract in the RNA of the RNA-DNA hybrid. Using biochemical approaches, and cryo-electron microscopy, this study demonstrates that the RNA exit channel widens to accommodate RNA hairpin formation. This expansion causes a mobile portion of RNAP to rotate relative to its static portion, adopting an inactive "swiveled state" that halts nucleotide incorporation. Although certain transcription factors can rescue swiveled and paused RNAP, this work reveals that NusA stabilizes RNAP in its swiveled and paused state. In contrast, NusG shows species-specific effects: in Mycobacterium tuberculosis, it stabilizes the swiveled and paused state, whereas in Escherichia coli, it reverses the swivel, promoting RNAP elongation. For RNAP paused at intrinsic terminators, this study shows that RNAP must rewind the transcription bubble to allow the terminator hairpin to fully form. A complete terminator hairpin triggers the release of RNA and then DNA. These findings define a fundamental structural mechanism of bacterial RNAP during hairpin-stabilized pausing and intrinsic termination, providing a broader understanding of transcription regulation.
Author: Smarajit Mondal
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Transcription in bacteria is regulated at the level of initiation, elongation and termination. Although the regulation of transcriptional initiation is well studied, the regulation of elongation and termination are not well understood. This thesis focuses on understanding the role of NusA on intrinsic termination and the role of NusG on RNA polymerase pausing using genomic, biochemical and computational analyses. Tight regulation of transcription termination is required to maintain proper levels of gene expression in bacteria, because termination failure abolishes operon boundaries, leading to misregulation of downstream genes. NusA is a negative transcription elongation factor that was known to cause a slight stimulation of termination at intrinsic terminators in vitro, but its impact on termination and global gene expression in vivo was not known. In this thesis, I describe the mapping of intrinsic terminators genome wide in B subtilis and measure the effect of NusA on the efficiency of these terminators in vivo using a novel high resolution 3' end-mapping technique coupled with mRNA profiling. Based on these studies, I report the existence of a subclass of previously unidentified pseudo-intrinsic terminators that are dependent on NusA for termination. Sequence comparison of different terminators reveals that weak hairpins and/or distal U-tract interruptions favors NusA-dependent termination, supporting a model in which NusA assists hairpin folding and slows down RNA polymerase near the termination window. These studies also revealed that readthrough of NusA-dependent terminators increases transcription of genes related to replication and DNA metabolism, suggesting a role of NusA in maintaining genome stability. I further show that nusA is autoregulated by a transcription attenuation mechanism that does not rely on antiterminator structures to prevent termination. Instead, NusA-stimulated termination in its 5'UTR dictates the extent of transcription into the operon. Another major focus of this thesis is to understand the regulation of transcription elongation by NusG-stimulated pausing of RNA polymerase. NusG is a positive elongation factor in E. coli that accelerates transcription by reducing the dwell time of RNA polymerase at pause sites. In B. subtilis, NusG stimulates pausing at positions U107 and U144 in the trp-leader transcript. NusG-stimulated pausing at U144 requires a short sequence in the non-template DNA strand and participates in the TRAP-dependent translation repression mechanism. In this thesis, I report the characterization of the NusG-stimulated U107 pause signal and show that disruption of the NusG recognition motif dramatically reduces pausing. These results suggest a mechanism in which RNA polymerase pausing at this site participates in the transcription attenuation mechanism by increasing additional time for TRAP binding to the nascent transcript.
Author: Tilman Heise
Publisher:
ISBN: 9781071602317
Category : Human genetics
Languages : en
Pages : 314
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Book Description
This book provides a wide spectrum of methods to study RNA chaperones in vitro, at the single molecule level, and protocols useful for cell-based assays. Beginning with a section on a number of bacterial proteins for study, the volume also explores proteins from eukaryotic cells and how to delve into the complex interactions between RNA chaperones and the folding and unfolding of proteins. 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, RNA Chaperones: Methods and Protocols serves as an ideal guide for scientists and students interested in RNA biology and RNA chaperones. Chapter 3 is available Open Access under a CC-BY 4.0 license via link.springer.com.
Author: Xieyang Guo
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, I present single-particle electron cryo-microscopy (cryo-EM) reconstructions of NusA bound to paused elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor.
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 276
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During RNA synthesis, RNA polymerase (RNAP) can temporally halt nucleotide addition by pausing. Transcriptional pausing plays a central role in regulation of transcription and may involve conformational changes in RNAP due to interactions of RNAP with intrinsic signals encoded in DNA and RNA, changes in translocation register, or both. However, basic mechanisms of transcriptional pausing and the role of RNAP translocation in regulation of transcript elongation are poorly understood. Here, we present work to investigate pause mechanisms, specifically how the pause signal is integrated/transmitted from the RNA exit channel of the enzyme to the enzymefs active site, and the contributions of RNAP translocation to pausing. We determined that the identity of RNA:DNA nucleotides in the active site are strong determinants of translocation bias, with the 3L RNA nucleotide favoring the pretranslocated state in the order U>C>A>G. Transcript elongation in bacteria is regulated in part by structures that form in the nascent RNA transcript and interact with RNAP in the RNA exit channel of the enzyme. A pause RNA hairpin structure makes direct contact with the flap domain of the RNAP f subunit, and slows nucleotide addition by factors of 10-20. We investigated the effects of different length and type of duplexes in the RNAP exit channel on pausing and response to regulators. We show that 8-bp RNA duplexes can stimulate pausing and those with less than 8-bp duplexes do not give full hairpin effect. We also investigated how regulatory communication between the formation of secondary structure in the RNA exit channel and the active site of RNAP modulate the catalytic center function. The opening of the RNAP clamp has been proposed to occur during pausing and stabilize the pause state. Our findings indicate that the formation of the paused 8-bp RNA:RNA duplex in the RNA exit channel causes movement of RNAPfs clamp and the flap domains, which in turn inhibits RNAP translocation and the catalysis of rapid nucleotide transfer. Transcription factors like NusA and RfaH tune and provide additional function to this communication network by affecting the clamp movements, translocation, or both functions.
Author: Rachel Anne Mooney
Publisher:
ISBN:
Category :
Languages : en
Pages : 302
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Author: Walid El-Sharoud
Publisher: Springer Science & Business Media
ISBN: 3540749217
Category : Science
Languages : en
Pages : 377
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Book Description
The application of new molecular methodologies in the study of bacterial behavior and cell architecture has enabled new revolutionary insights and discoveries in these areas. This new text presents recent developments in bacterial physiology that are highly relevant to a wide range of readership including those interested in basic and applied knowledge. Its chapters are written by international scientific authorities at the forefront of the subject. The value of this recent knowledge in bacterial physiology is not only restricted to fundamental biology. It also extends to biotechnology and drug-discovery disciplines.
Author: Victor A. Bloomfield
Publisher: Sterling Publishing Company
ISBN: 9780935702491
Category : Science
Languages : en
Pages : 854
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Book Description
Providing a comprehensive account of the structures and physical chemistry properties of nucleic acids, with special emphasis on biological function, this text has been organized to meet the needs of those who have only a basic understanding of physical chemistry and molecular biology.
Author: Uldis N. Streips
Publisher: John Wiley & Sons
ISBN: 0471461083
Category : Science
Languages : en
Pages : 673
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Book Description
In accordance with its predecessor, the completely revised and expanded Second Edition of Modern Microbial Genetics focuses on how bacteria and bacteriophage arrange and rearrange their genetic material through mutation, evolution, and genetic exchange to take optimal advantage of their environment. The text is divided into three sections: DNA Metabolism, Genetic Response, and Genetic Exchange. The first addresses how DNA replicates, repairs itself, and recombines, as well as how it may be manipulated. The second section is devoted to how microorganisms interact with their environment, including chapters on sporulation and stress shock, and the final section contains the latest information on classic exchange mechanisms such as transformation and conjugation. Chapters include: * Gene Expression and Its Regulation * Single-Stranded DNA Phages * Genetic Tools for Dissecting Motility and Development of Myxococcus xanthus * Molecular Mechanism of Quorum Sensing * Transduction in Gram-Negative Bacteria * Genetic Approaches in Bacteria with No Natural Genetic Systems The editors also cultivate an attention to global regulatory systems throughout the book, elucidating how certain genes and operons in bacteria, defined as regulons, network and cooperate to suit the needs of the bacterial cell. With clear appreciation for the impact of molecular genomics, this completely revised and updated edition proves that Modern Microbial Genetics remains the benchmark text in its field.
Author: Nancy Craig
Publisher: Oxford University Press
ISBN: 0199658579
Category : Science
Languages : en
Pages : 945
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Book Description
The biological world operates on a multitude of scales - from molecules to tissues to organisms to ecosystems. Throughout these myriad levels runs a common thread: the communication and onward passage of information, from cell to cell, from organism to organism and ultimately, from generation to generation. But how does this information come alive to govern the processes that constitute life? The answer lies in the molecular components that cooperate through a series of carefully-regulated processes to bring the information in our genome to life. These components and processes lie at the heart of one of the most fascinating subjects to engage the minds of scientists today: molecular biology. Molecular Biology: Principles of Genome Function, Second Edition, offers a fresh approach to the teaching of molecular biology by focusing on the commonalities that exist between the three kingdoms of life, and discussing the differences between the three kingdoms to offer instructive insights into molecular processes and components. This gives students an accurate depiction of our current understanding of the conserved nature of molecular biology, and the differences that underpin biological diversity. Additionally, an integrated approach demonstrates how certain molecular phenomena have diverse impacts on genome function by presenting them as themes that recur throughout the book, rather than as artificially separated topics As an experimental science, molecular biology requires an appreciation for the approaches taken to yield the information from which concepts and principles are deduced. Experimental Approach panels throughout the text describe research that has been particularly valuable in elucidating difference aspects of molecular biology. Each panel is carefully cross-referenced to the discussion of key molecular biology tools and techniques, which are presented in a dedicated chapter at the end of the book. Molecular Biology further enriches the learning experience with full-color artwork, end-of-chapter questions and summaries, suggested further readings grouped by topic, and an extensive glossary of key terms. Features: A focus on the underlying principles of molecular biology equips students with a robust conceptual framework on which to build their knowledge An emphasis on their commonalities reflects the processes and components that exist between bacteria, archae, and eukaryotes Experimental Approach panels demonstrate the importance of experimental evidence by describing research that has been particularly valuable in the field
