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Biochemical And Genetic Analysis Of Interactions Between The Escherichia Coli Transcription Activator Rhas And Rna Polymerase
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Book Synopsis Biochemical and Genetic Analysis of Interactions Between the Escherichia Coli Transcription Activator RhaS and RNA Polymerase by : Visnja Jevtic
Download or read book Biochemical and Genetic Analysis of Interactions Between the Escherichia Coli Transcription Activator RhaS and RNA Polymerase written by Visnja Jevtic and published by . This book was released on 2004 with total page 250 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Molecular Interactions Between the Transcription Factor Crl and Sigma S RNA Polymerase Holoenzyme in Escherichia Coli by :
Download or read book Molecular Interactions Between the Transcription Factor Crl and Sigma S RNA Polymerase Holoenzyme in Escherichia Coli written by and published by . This book was released on 2013 with total page 586 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bacteria must change their cellular composition in response to changing environmental conditions and stress. They implement these changes by altering gene expression and/or protein activity. Central to changes in gene expression during stress responses is regulation of transcription. In bacteria, transcription is directed by a suite of RNA polymerase (RNAP) holoenzymes, comprised of a core enzyme, possessing the catalytic activity for RNA synthesis, and one of a collection of transcription initiation factors, known as Ï3 factors, which recognizes promoter DNA sequences upstream of genes. Increasing the formation of a particular type of RNAP holoenzyme by switching the Ï3 factor will lead to expression of its cognate regulon. My thesis research focuses on deciphering the complex molecular interactions between the transcription factor Crl and the general stress response sigma factor, Sigma S, in order to understand Escherichia coli's response to stress at the molecular level. Crl is a small (133 amino acids in E. coli) protein that stimulates transcription mediated by Sigma S. At the outset of my research, little was known about how Crl interacts with the transcription machinery to stimulate Sigma S-mediated transcription. I used a combination of biochemical, genetic, and molecular biological techniques in vitro and in vivo to identify key interactions and gain insight into the mechanism of transcriptional regulation by Crl. I identify a key binding determinant (the DPE motif) in Sigma S conserved domain 2 underlying its specific recognition by Crl. I demonstrate directly that Crl requires this determinant for positive regulation of Sigma S-mediated transcription and to enhance Sigma S holoenzyme formation. I made the primary Sigma factor recognizable by Crl by substitution of the DPE motif along with deletion of a large non-conserved region (NCR). I also localized the area of Crl required for the interaction with Sigma S to Crl's conserved central cleft. Finally, I identified an interaction between the Beta prime subunit of core RNAP and Crl, which occurs only in the context of the holoenzyme, that has begun to clarify the mechanism by which Crl functions as a Sigma S-specific RNAP holoenzyme assembly factor.
Book Synopsis Genetic Analysis of RNA Polymerase/transcriptional Activator Interactions at ThepepT Promoter of S. Typhimurium by : Mary-Jane Lombardo
Download or read book Genetic Analysis of RNA Polymerase/transcriptional Activator Interactions at ThepepT Promoter of S. Typhimurium written by Mary-Jane Lombardo and published by . This book was released on 1995 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The role of the alpha subunit of RNA polymerase (encoded by rpoA) in transcriptional activation by the global regulatory protein FNR has been investigated using a genetic approach. We have identified five rpoA mutations which decrease the FNR-dependent anaerobic induction of the pepT gene of S. typhimurium. Each of these mutations (G311E, G311R, L289F, R317H, W320ter) affects an amino acid in the C-terminal region of the alpha subunit. Phenotypic characterization shows that four of the five mutations have relatively specific effects on positively regulated transcription of the pepT gene and other FNR-dependent genes. We propose that the C-terminal region of the alpha subunit is involved in direct interactions with FNR. We reasoned that suppressor analysis might be a useful approach to identifying regions of FNR that are involved in interactions with RNA polymerase. Reversion of the G311E and G311R mutations led to the isolation of six mutations (E47K, T80I, A172V, A172T, V208A, and V208G) in the transcriptional activator FNR which restore or partially restore anaerobic induction of pepT in the presence of each of the five rpoA mutations. A172 and V208 both lie in the DNA binding domain of FNR suggesting that the mutations at these positions might restore activation by affecting DNA binding directly or indirectly. The T80I and E47K mutations lie elsewhere in the protein and are unlikely to affect DNA binding. We suggest that the T80 and E47 regions of FNR might be involved in direct interactions between FNR and the alpha subunit or another subunit of RNA polymerase. In addition, we have characterized the pepT promoter region. Mutational studies and primer extension analysis demonstrate that pepT is transcribed from two promoters, an FNR-dependent, anaerobically induced promoter (P1), and a constitutive promoter (P2) conferring low level expression. A mutant P1 promoter containing a consensus-10 sequence was shown to be regulated by CRP-cAMP in addition to FNR. The potABCD operon was shown to be divergently transcribed from pepT, and the potABCD promoter was identified.
Book Synopsis Characterization of "on DNA" and "off DNA" Interactions Between Transcriptional Activator SoxS and RNA Polymerase of Escherichia Coli by : Muhammad Ammar Zafar
Download or read book Characterization of "on DNA" and "off DNA" Interactions Between Transcriptional Activator SoxS and RNA Polymerase of Escherichia Coli written by Muhammad Ammar Zafar and published by . This book was released on 2009 with total page 432 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis The Interactions of Escherichia Coli RNA Polymerase with DNA by : Paul Melancon
Download or read book The Interactions of Escherichia Coli RNA Polymerase with DNA written by Paul Melancon and published by . This book was released on 1983 with total page 528 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Studies on the Conformation of Escherichia Coli Catabolite Activator Protein and RNA Polymerase when They Interact with Promoter DNA by : Mark Henry Sinton
Download or read book Studies on the Conformation of Escherichia Coli Catabolite Activator Protein and RNA Polymerase when They Interact with Promoter DNA written by Mark Henry Sinton and published by . This book was released on 1993 with total page 468 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Interactions Between Phage T7 Early Promoters and E. Coli RNA Polymerase by : Tao-shih Hsieh
Download or read book Interactions Between Phage T7 Early Promoters and E. Coli RNA Polymerase written by Tao-shih Hsieh and published by . This book was released on 1976 with total page 200 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis The Mechanism of Interaction of E. Coli RNA Polymerase with Bacteriophage and Bacterial Promoters by : Sigrid Leirmo
Download or read book The Mechanism of Interaction of E. Coli RNA Polymerase with Bacteriophage and Bacterial Promoters written by Sigrid Leirmo and published by . This book was released on 1989 with total page 324 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Studies on the Interaction of E. Coli RNA Polymerase with Lactose Promoter DNA by : Donald Duane Lorimer
Download or read book Studies on the Interaction of E. Coli RNA Polymerase with Lactose Promoter DNA written by Donald Duane Lorimer and published by . This book was released on 1989 with total page 260 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Studies of Interaction Between the Heat Shock Sigma Factor, O23, and Core RNA Polymerase in Escherichia Coli by : Daniel M. Joo
Download or read book Studies of Interaction Between the Heat Shock Sigma Factor, O23, and Core RNA Polymerase in Escherichia Coli written by Daniel M. Joo and published by . This book was released on 1997 with total page 308 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Interactions of the Nascent Transcript with the E. Coli RNA Polymerase in Elongation Complexes by : Sandra Milan
Download or read book Interactions of the Nascent Transcript with the E. Coli RNA Polymerase in Elongation Complexes written by Sandra Milan and published by . This book was released on 1995 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Local- and Genome-scale Study of the Interplay Between Escherichia Coli RNA Polymerase and Nucleoid-associated Proteins by : Erik Dean Jessen
Download or read book Local- and Genome-scale Study of the Interplay Between Escherichia Coli RNA Polymerase and Nucleoid-associated Proteins written by Erik Dean Jessen and published by . This book was released on 2018 with total page 185 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bacterial transcription, once thought to be organized into discrete, largely non-overlapping units, has been revealed by deep cDNA sequencing to generate ubiquitous, overlapping, sense and antisense RNAs, many of which are noncoding. The function of the extensive antisense transcription of bacterial genes is controversial, and unclear in lieu of mechanistic dissection. We report here characterization of a model antisense transcription unit (bglAS) in the cryptic, H-NS-silenced b-glucoside utilization operon of E. coli K-12 (bglGFBH). bglAS was discovered because inhibition of Rho greatly increased its level and length. We created bglAS- alleles with little or no effect on bglF encoded in the sense strand by disabling the bglAS promoter with base substitutions. Although bglAS exists in a small H-NS-free island in the otherwise H-NS-coated bgl operon, the H-NS distribution is unchanged in bglAS- strains. However, when bglGFBH sense transcription was activated, bglAS decreased b-glucoside-induction of bgl gene expression via BglG-mediated antitermination of the bgl operon attenuators. Using a time series of ChIP-chip, we found that RNA polymerase progression through bglGFBH is hindered by bglAS transcription. In contrast, overexpression of bglAS RNA in trans had no effect on bgl operon induction, supporting bglAS function by transcriptional interference with bglGFBH expression. The bglAS promoter was upregulated by nitrite, consistent with bioinformatic detection of adjacent NarL/P binding sites and suggesting potential bglAS function as an environmental modulator. The proximity of bglAS to the surrounding H-NS filaments led us to investigate the interactions between transcription and H-NS. Consistent with the inability of transcription to affect H-NS binding patterns at the bgl operon, the genome-scale H-NS distribution exhibited minimal change when all transcription was inhibited with rifampicin. However, deletion of H-NS and StpA, an H-NS paralog, resulted in increased progression of RNAP along active, H-NS bound transcription units. Analyzing NET-seq data suggested that H-NS inhibits RNAP progression by promoting pausing of RNAP at non-canonical pause sequences. In contrast to H-NS, the nucleoid-associated protein HU was found to have a distribution pattern mirroring highly active transcription units. Upon further investigation, HU most closely correlated with the presence of R-loops, stable RNA:DNA hybrids external of RNAP. R-loop levels are independent of the presence of HU, suggesting HU is not involved in the resolution of R-loops. Instead, deletion of HU confers sensitivity to reactive oxygen species, and we propose a model by where HU binds to the areas around R-loops to protect DNA from mutagenesis. The combined studies presented here are a significant advancement in our understanding of the function of antisense transcripts and the interaction between transcription and nucleoid-associated proteins.
Book Synopsis Investigation of the Topological Interactions Between Escherichia Coli Transcripton Factor Rho and RNA by : Brandt R. Burgess
Download or read book Investigation of the Topological Interactions Between Escherichia Coli Transcripton Factor Rho and RNA written by Brandt R. Burgess and published by . This book was released on 2001 with total page 266 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Genetic Analysis of the [beta] Subunit of Escherichia Coli RNA Polymerase by : Patricia L. Tavormina
Download or read book Genetic Analysis of the [beta] Subunit of Escherichia Coli RNA Polymerase written by Patricia L. Tavormina and published by . This book was released on 1994 with total page 340 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Protein-protein Interactions Within Escherichia Coli RNA Polymerase Mapped by Artificial Iron-dependent Proteases by : Saul Andrew Datwyler
Download or read book Protein-protein Interactions Within Escherichia Coli RNA Polymerase Mapped by Artificial Iron-dependent Proteases written by Saul Andrew Datwyler and published by . This book was released on 2001 with total page 310 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Multiplexed Approaches to Characterize Sequence-Function Relationships of Escherichia Coli Promoters by : Guillaume Urtecho
Download or read book Multiplexed Approaches to Characterize Sequence-Function Relationships of Escherichia Coli Promoters written by Guillaume Urtecho and published by . This book was released on 2020 with total page 257 pages. Available in PDF, EPUB and Kindle. Book excerpt: Despite decades of intense genetic, biochemical, and evolutionary characterizations of promoters in Escherichia coli, we still lack the basic ability to identify which genomic sequences represent promoters as well as the transcriptional activity of these sequences. Furthermore, roughly two-thirds of the 2,565 reported E. coli operons do not contain any transcription factor binding site annotations, highlighting our lack of understanding the regulation of these essential genetic components. In my thesis work, I sought to fill this lack of understanding by 1) Identifying promoters in the E. coli genome, 2) Discovering the regulatory elements within these promoters that encode their activity, and 3) Characterizing the combinatorial interactions between promoter regulatory elements to learn how they cooperatively determine expression. During the early stages of my graduate work I developed a genomically-encoded massively parallel reporter assay to measure promoter activity of hundreds of thousands of DNA sequences simultaneously in E. coli. In Chapter 3 we used this technology to perform the first full characterization of autonomous promoter activity in E. coli, measuring promoter activity of >300,000 sequences spanning the entire genome and precisely mapping 2,859 promoters active in rich media. After identifying promoters in E. coli, we sought to deconstruct these sequences to learn how they encoded promoter regulation. We performed a scanning mutagenesis of these discovered promoters and identified the sequence motifs and elements within these regions, providing insights into the regulation of 1,158 E. coli operons. Overall, we generated a genome-wide atlas of promoters in E. coli as well as a rich dataset for future computational modeling projects to dissect the relationship between DNA sequence and promoter function. Concurrent with our work to identify promoters and their regulatory sequence elements, we also sought to learn how combinations of these regulatory sequences cooperatively determine expression. Basic promoters are composites of multiple discrete sequence elements recognized by RNA polymerase (RNAP). While it is known that the overall affinity of RNAP to these elements determines the strength of the promoter it has been unclear how combinations of these motifs collectively determine promoter activity. To explore this, in Chapter 4 we measured the activity of over 10,000 synthetic promoters composed of different combinations of RNAP binding sites that spanned a range of affinities. We learned that synergistic and other non-linear interactions between RNAP binding sites are responsible for a significant proportion of variance in promoter activity and by capturing these interactions in a statistical model, we can predict the activity of promoters with over 95% accuracy. Furthermore, we discovered the novel phenomenon that promoters composed of the strongest sequence elements function poorer than expected, as their overpowering binding affinities prevent RNAP from escaping to perform transcription. In Chapter 5, we expand on this analysis to study combinatorial interactions in the context of repression by LacI. By studying 8,269 lacUV5 promoter variants composed of different combinations of RNAP and LacI repressor sites, we are able to study the interactions between these sites in a variety of binding site arrangements. This work revealed the principle relationships between RNAP and repressors as well as provided insight in how to tune repressor binding site affinities in order to maximize inducibility of promoters for synthetic biology applications. These projects have greatly expanded what is known about the native E. coli promoter landscape, revealed insights on how promoter organization influences their roles, and deconstructed the interactions between sequence elements that compose promoters. In addition to the knowledge of E. coli promoter regulation, the technologies and methodologies we developed in this work can be used to characterize virtually any genetically tractable bacteria.
Download or read book The Operon written by Jeffrey H. Miller and published by . This book was released on 1980 with total page 488 pages. Available in PDF, EPUB and Kindle. Book excerpt:
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