Elucidating the Contributions of Promoter Sequence and (p)ppGpp Binding Sites on RNAP to Transcription Regulation in Escherichia Coli PDF Download
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Author: Rachel Isabelle Salemi
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Bacteria must respond quickly to stressful environmental stimuli and nutrient deprivation in order to survive. Conserved across the bacterial domain are the intracellular stress signaling molecules ppGpp and pppGpp, referred to here as (p)ppGpp for brevity. In Escherichia coli and other gammaproteobacteria, (p)ppGpp binds directly to RNA polymerase (RNAP) to affect the transcription of hundreds of genes. Depending on the intrinsic promoter kinetics, transcription from promoters will be inhibited, activated, or unaffected by (p)ppGpp. Because (p)ppGpp affects the kinetics of transcription during the isomerization steps of open complex formation, determining promoter sequences that result in regulation by (p)ppGpp is extremely challenging as there are many sequences that can result in the same kinetic profile. To address this issue, we compiled a promoter dataset of over 1000 promoters and compared promoters that were inhibited, unaffected, or activated by (p)ppGpp to determine sequences that may be responsible for regulation. We find that promoters with specific bases at the -9 and -8 positions, -5 position, and three bases upstream of the transcription start site correlate with regulation via (p)ppGpp. In addition to better understanding the promoter sequences for regulation by (p)ppGpp, we explored the purpose of the two (p)ppGpp binding sites on RNAP. (p)ppGpp binds to two sites on RNAP: site 1 at the interface of [omega] and [beta]' subunits, and site 2 at the interface of DksA and [beta]'. Large phenotypic effects are associated with site 2 both in vitro and in vivo, but the function of site 1 has remained a mystery. Despite the lack of obvious function, site 1 is well-conserved throughout gammaproteobacteria and binds with similar affinity to (p)ppGpp as site 2. Using RNA-sequencing before and after (p)ppGpp induction in strains lacking either site 1 or site 2, we confirm that site 1 is capable only of inhibition and not of activation, and that site 2 performs the majority of gene regulation on a short time scale after induction of (p)ppGpp. We also find that in the conditions we tested, during steady state growth strains lacking site 1 are unable to properly express genes from the [sigma]S regulon and exhibit decreased [sigma]S protein levels compared to strains containing site 1. Additionally, strains lacking site 1 were sensitive to low pH, while strains containing site 1 regardless of the presence of site 2 survived exposure to low pH. Together, this work provides a new perspective on both promoter analysis for promoters regulated by (p)ppGpp and the function of site 1 through implementing genome-wide approaches.
Author: Rachel Isabelle Salemi
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Bacteria must respond quickly to stressful environmental stimuli and nutrient deprivation in order to survive. Conserved across the bacterial domain are the intracellular stress signaling molecules ppGpp and pppGpp, referred to here as (p)ppGpp for brevity. In Escherichia coli and other gammaproteobacteria, (p)ppGpp binds directly to RNA polymerase (RNAP) to affect the transcription of hundreds of genes. Depending on the intrinsic promoter kinetics, transcription from promoters will be inhibited, activated, or unaffected by (p)ppGpp. Because (p)ppGpp affects the kinetics of transcription during the isomerization steps of open complex formation, determining promoter sequences that result in regulation by (p)ppGpp is extremely challenging as there are many sequences that can result in the same kinetic profile. To address this issue, we compiled a promoter dataset of over 1000 promoters and compared promoters that were inhibited, unaffected, or activated by (p)ppGpp to determine sequences that may be responsible for regulation. We find that promoters with specific bases at the -9 and -8 positions, -5 position, and three bases upstream of the transcription start site correlate with regulation via (p)ppGpp. In addition to better understanding the promoter sequences for regulation by (p)ppGpp, we explored the purpose of the two (p)ppGpp binding sites on RNAP. (p)ppGpp binds to two sites on RNAP: site 1 at the interface of [omega] and [beta]' subunits, and site 2 at the interface of DksA and [beta]'. Large phenotypic effects are associated with site 2 both in vitro and in vivo, but the function of site 1 has remained a mystery. Despite the lack of obvious function, site 1 is well-conserved throughout gammaproteobacteria and binds with similar affinity to (p)ppGpp as site 2. Using RNA-sequencing before and after (p)ppGpp induction in strains lacking either site 1 or site 2, we confirm that site 1 is capable only of inhibition and not of activation, and that site 2 performs the majority of gene regulation on a short time scale after induction of (p)ppGpp. We also find that in the conditions we tested, during steady state growth strains lacking site 1 are unable to properly express genes from the [sigma]S regulon and exhibit decreased [sigma]S protein levels compared to strains containing site 1. Additionally, strains lacking site 1 were sensitive to low pH, while strains containing site 1 regardless of the presence of site 2 survived exposure to low pH. Together, this work provides a new perspective on both promoter analysis for promoters regulated by (p)ppGpp and the function of site 1 through implementing genome-wide approaches.
Author: Patricia Sánchez-Vázquez
Publisher:
ISBN:
Category :
Languages : en
Pages : 313
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Book Description
Upon amino acid starvation of Escherichia coli, the second messenger nucleotide ppGpp dramatically alters gene expression in order to adjust cellular metabolism to the nutritional conditions. ppGpp specifically regulates transcription by interacting directly with RNA polymerase (RNAP) at two sites identified in vitro. I created strains with chromosomal mutations in the residues identified in vitro, coding for residues needed for ppGpp binding to RNAP. I used recombineering combined with CRISPR-Cas9 selection followed by whole genome sequencing to clone and verify the binding site mutant strains created. The strains were used first to evaluate the physiological significance of ppGpp binding sites 1 and 2, and second as tools for separating the transcriptional effects of ppGpp binding to RNAP vs other potential targets. To study the direct genome-wide transcriptional effects of ppGpp, I performed RNA-seq analysis on cells in which ppGpp was induced without introducing a nutrient starvation, and we compared effects of ppGpp induction in cells lacking the ppGpp binding sites on RNAP rather than by eliminating the regulators themselves. The results identify a much larger number of ppGpp-responsive targets than previous studies and show that all transcriptional responses result from binding of ppGpp to RNAP rather than to other proteins. In vitro transcription analysis of more than 100 promoters from the in vivo dataset unequivocally identified a large collection of directly regulated promoters, facilitating comparison of the sequences from directly inhibited, activated, and unaffected promoters. This analysis suggests DNA sequence features that contribute to promoter specificity and to the mechanism of the stringent response. Together, the results presented here expand on the knowledge of regulatory targets and the mechanism of ppGpp action.
Author: Sizhe Qiu
Publisher:
ISBN:
Category :
Languages : en
Pages : 31
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Book Description
The transcriptional regulatory network (TRN) of E. coli MG1655 contains thousands of regulatory interactions between transcription factors, sigma factors and promoter sequences of genes. The membership of a regulon, in other words whether a gene is regulated by a transcription factor, has a basis in the gene's promoter. To determine the TRN, there are two primary families of methods: bottom-up identification of binding sites via methods such as ChIP-seq experiments, and top-down inference of the TRN via expression analysis methods such as independent component analysis (ICA). In this work, the promoter sequence of each gene was utilized to predict regulons with machine learning. Certain promoter features, such as TF binding site motif scores, DNA shape features, and sigma factor-related features were found to be essential to predict whether a gene is regulated by particular transcription factors. ICA and ChIP TRNs were compared to investigate factors underlying their difference, and two case studies were carried out. An FNR case study showed that ICA regulons fractionate a large ChIP regulon into several regulation types. An ArcA case study demonstrated that the ICA TRN captures diversity in binding sites' architecture. In general, through comparison, ICA TRN extracts genes of strong regulation activity from ChIP TRN. We then expanded this analysis to understand differences in the regulons of multiple strains of E. coli. A pan-regulon of Fur was reconstructed with unique, accessory and core regulons annotated. We found that genes in the core regulon have stronger regulation activity than genes in the unique regulon. Additionally, it was also found that the motif score and helix twist of DNA sequence were both significant indicators of Fur ChIP-exo peak heights, represented by S/N ratios. This study was a meaningful application of machine learning on biological problems that probed biophysical factors underlying omics data, giving directions for the genome design in synthetic biology aiming to control the phenotype by TRN tuning.
Author: Wolfram Saenger
Publisher: Springer Science & Business Media
ISBN: 1461251907
Category : Science
Languages : en
Pages : 574
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Book Description
New textbooks at all levels of chemistry appear with great regularity. Some fields like basic biochemistry, organic reaction mechanisms, and chemical ther modynamics are well represented by many excellent texts, and new or revised editions are published sufficiently often to keep up with progress in research. However, some areas of chemistry, especially many of those taught at the grad uate level, suffer from a real lack of up-to-date textbooks. The most serious needs occur in fields that are rapidly changing. Textbooks in these subjects usually have to be written by scientists actually involved in the research which is advancing the field. It is not often easy to persuade such individuals to set time aside to help spread the knowledge they have accumulated. Our goal, in this series, is to pinpoint areas of chemistry where recent progress has outpaced what is covered in any available textbooks, and then seek out and persuade experts in these fields to produce relatively concise but instructive introductions to their fields. These should serve the needs of one semester or one quarter graduate courses in chemistry and biochemistry. In some cases the availability of texts in active research areas should help stimulate the creation of new courses. CHARLES R. CANTOR New York Preface This monograph is based on a review on polynucleotide structures written for a book series in 1976.
Author: Phoebe A. Rice
Publisher: Royal Society of Chemistry
ISBN: 0854042725
Category : Science
Languages : en
Pages : 417
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Book Description
This book provides both in-depth background and up-to-date information in this area. The chapters are organized by general themes and principles, written by experts who illustrate topics with current findings. Topics covered include: - the role of ions and hydration in protein-nucleic acid interactions - transcription factors and combinatorial specificity - indirect readout of DNA sequence - single-stranded nucleic acid binding proteins - nucleic acid junctions and proteins, - RNA protein recognition - recognition of DNA damage. It will be a key reference for both advanced students and established scientists wishing to broaden their horizons.
Author: Thomas J. Silhavy
Publisher:
ISBN:
Category : Science
Languages : en
Pages : 332
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Author:
Publisher:
ISBN:
Category : Medicine
Languages : en
Pages : 2084
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Book Description
Vols. for 1963- include as pt. 2 of the Jan. issue: Medical subject headings.
Author: Sang Yup Lee
Publisher: Springer Science & Business Media
ISBN: 1402093942
Category : Science
Languages : en
Pages : 471
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Book Description
Systems biology is changing the way biological systems are studied by allowing us to examine the cell and organism as a whole. Systems biotechnology allows optimal design and development of upstream to downstream bioprocesses by taking a systems-approach. E. coli has been a model organism for almost all biological and biotechnological studies. This book brings together for the first time the state-of-the-art reviews by the world-leading experts on systems biology and biotechnological applications of E. coli. The topics covered include genomics and functional genomics, resources for systems biology, network analysis, genome-scale metabolic reconstruction, modelling and simulation, dynamic modelling and simulation, systems-level analysis of evolution, plasmids and expression systems, protein synthesis, production and export, engineering the central metabolism, synthetic biology, and systems metabolic engineering of E. coli. This book provides readers with guidance on how a complex biological system can be studied using E. coli as a model organism. It also presents how to perform synthetic biology and systems metabolic engineering studies on E. coli with successful examples, the approaches of which can be extended to other organisms. This book will be a complete resource for anyone interested in systems biology and biotechnology.
Author: Gisela Storz
Publisher: John Wiley & Sons
ISBN: 1683672941
Category : Medical
Languages : en
Pages : 1065
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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: John W. Pelley, PhD
Publisher: Elsevier Health Sciences
ISBN: 0323074464
Category : Medical
Languages : en
Pages : 254
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Book Description
Effectively merge basic science and clinical skills with Elsevier's Integrated Review Biochemistry, by John W. Pelley, PhD. This concise, high-yield title in the popular Integrated Review Series focuses on the core knowledge in biochemistry while linking that information to related concepts from other basic science disciplines. Case-based questions at the end of each chapter enable you to gauge your mastery of the material, and a color-coded format allows you to quickly find the specific guidance you need. Online access via www.studentconsult.com - included with your purchase - allows you to conveniently access the book's complete text and illustrations online as well as relevant content from other Student Consult titles. This concise and user-friendly reference provides crucial guidance for the early years of medical training and USMLE preparation. Spend more time reviewing and less time searching thanks to an extremely focused, "high-yield" presentation. Gauge your mastery of the material and build confidence with both case-based, andUSMLE-style questions that provide effective chapter review and quick practice for your exams. Access the full contents online at www.studentconsult.com where you'll find the complete text and illustrations, "Integration Links" to bonus content in other Student Consult titles, an interactive community center with a wealth of additional resources, and much more! Grasp and retain vital concepts more easily thanks to a color-coded format, succinct,text, key concept boxes, and dynamic illustrations that facilitate learning in a highly visual approach. Effectively review for problem-based courses with the help of text boxes that help you clearly see the clinical relevance of the material. Great for visual learners!
