Regulation of Transcription by Nusa And Nusg in Bacillus Subtilis PDF Download
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Author: Smarajit Mondal
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Languages : en
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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: Smarajit Mondal
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
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: Zachary Mandell
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Category :
Languages : en
Pages : 0
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Gene expression is regulated at many levels, including both the transcription and decay of RNA. Transcription terminates at discrete locations across the genome. After release, the resultant transcripts are degraded. This thesis outlines recent studies that utilized biochemical, molecular, genetic, genomic, physiological, and computational techniques to further our understanding of how transcription termination and RNA decay are regulated in the Gram+ organism Bacillus subtilis. Transcription termination is known to occur via two mechanisms in bacteria, intrinsic termination (also frequently referred to as Rho-independent, or factor-independent termination), and Rho-dependent termination. Based primarily on in vitro studies using Escherichia coli RNA polymerase, it was generally assumed that intrinsic termination and Rho-dependent termination are distinct mechanisms, and that the signals required for intrinsic termination are present primarily within the nascent RNA. In this dissertation, I detail experiments that I conducted in B. subtilis, which show that intrinsic termination in this organism is highly stimulated by NusA, NusG, and even Rho. In NusA-stimulated intrinsic termination, NusA facilitates the formation of weak terminator hairpins and compensates for distal U-rich tract interruptions. In NusG-stimulated intrinsic termination, NusG stabilizes a sequence-dependent pause at the point of termination, which extends the timeframe for RNA hairpins with weak terminal base pairs to form in either a NusA-stimulated or a NusA-independent fashion. Rho stimulates intrinsic termination by preventing the formation of antiterminator-like RNA structures that could otherwise compete with the terminator hairpin. Combined, NusA, NusG, and Rho stimulate approximately 97% of all intrinsic terminators in B. subtilis. Thus, the historical distinction between Rho-dependent and intrinsic termination is overly simplistic and needs to be modernized. Moreover, the general view that intrinsic termination is primarily a factor-independent process needs to be revised to account for recent findings. The B. subtilis genome encodes for four known 3' to 5' exoribonucleases; polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. In E. coli, PNPase functions as a member of a multi-protein complex dedicated to the decay of mRNA. This complex is known as the degradosome. One function of the degradosome is to ensure that PNPase remains physically associated with RhlB, an RNA helicase. Whether PNPase operates in the context of a degradosome in B. subtilis remains controversial. In this dissertation I describe experiments that I conducted in B. subtilis, showing that PNPase cooperates with CshA, an RNA helicase, to degrade particularly structured mRNAs on a genome-wide level. Moreover, I obtained evidence that this cooperation occurs independently of a degradosome. In addition, I detail experiments that point towards the presence of a fifth, as-yet unidentified, 3' exoribonuclease.
Author: Expery O. Omollo
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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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: Steven L. McKnight
Publisher:
ISBN:
Category : Genetic regulation
Languages : en
Pages : 656
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Author: Aleksandra Grylak-Mielnicka
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Bacterial transcription is a process in which the information encoded in DNA is transferred to messenger RNA (mRNA). During the final step of this process, transcription termination, mRNA is released and can be used for protein synthesis. One type of transcription termination described in bacteria is Rho-dependent termination. The role of Rho has been widely investigated in model Gram-negative bacterium, Escherichia coli in which Rho is essential an abundant protein. In contrast, the knowledge about Rho inbacteria in which it is not essential and is present in low amounts, i. e. Gram-positive Bacillus subtilis remains limited.To investigate the role of Rho in control of gene expression in B. subtilis several large-scale analysis were performed. In effect, a set of Rho-specific physical and functional interactions were established. Additionally, new phenotypes of rho-null mutant were described unraveling the role of Rho in control of different aspects of cell physiology.
Author: Elizabeth Jean Bailey
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Category :
Languages : en
Pages :
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Transcription and translation are essential cellular processes that are coupled in bacteria. Though it was well-known that the rate of translation matches the rate of transcription, only in 2010 did evidence suggest direct physical coupling between the transcribing RNA polymerase (RNAP) and the translating ribosome. Nuclear magnetic resonance spectroscopy data showed that the RNAP-binding, transcription factor NusG could bind to the small ribosomal subunit protein, S10, through its C-terminal domain, thus, suggesting a model in which NusG simultaneously binds the transcription and translation machineries. In Chapter Two, I describe my investigations of the mechanism through which NusG-mediated transcription-translation coupling is established in bacteria, and how this coupling is regulated during gene expression. Specifically, I employed cell extract-based luciferase assays and purified C-terminal NusG mutants to show that the NusG N-terminal domain (NTD) and NusG F165A both inhibit transcription.
Author: Daniel Fernando Rojas Tapias
Publisher:
ISBN:
Category :
Languages : en
Pages : 398
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Author: Tilman Heise
Publisher:
ISBN: 9781071602317
Category : Human genetics
Languages : en
Pages : 314
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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: Gisela Storz
Publisher: John Wiley & Sons
ISBN: 1683670248
Category : Medical
Languages : en
Pages : 400
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Book Description
Revealing the many roles of RNA in regulating gene expression For decades after the discoveries of messenger RNA, transfer RNA, and ribosomal RNA, it was largely assumed that the role of RNA in the cell was limited to shuttling the genomic message, chaperoning amino acids, and toiling in the ribosomes. Eventually, hints that RNA molecules might have regulatory roles began to appear. With the advent of genomics and bioinformatics, it became evident that numerous other RNA forms exist and have specific functions, including small RNAs (sRNA), RNA thermometers, and riboswitches to regulate core metabolic pathways, bacterial pathogenesis, iron homeostasis, quorum sensing, and biofilm formation. All of these functions, and more, are presented in Regulating with RNA in Bacteria and Archaea, written by RNA biologists from around the globe. Divided into eight sections-RNases and Helicases, Cis-Acting RNAs, Cis Encoded Base Pairing RNAs, Trans-Encoded Base Pairing RNAs, Protein Titration and Scaffolding, General Considerations, Emerging Topics, and Resources-this book serves as an excellent resource for established RNA biologists and for the many scientists who are studying regulated cellular systems. It is no longer a fair assumption that gene expression regulation is the provenance of proteins only or that control is exerted primarily at the level of transcription. This book makes clear that regulatory RNAs are key partners along with proteins in controlling the complex interactions and pathways found within prokaryotes.
Author: Rajesh K. Gaur
Publisher: CRC Press
ISBN: 1420008706
Category : Science
Languages : en
Pages : 440
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New Findings Revolutionize Concepts of Gene FunctionEndogenous small RNAs have been found in various organisms, including humans, mice, flies, worms, fungi, and bacteria. Furthermore, it's been shown that microRNAs acting as cellular rheostats have the ability to modulate gene expression. In higher eukaryotes, microRNAs may regulate as much as 50 p
