Sigma 70-dependent RNA Polymerase Promoter-proximal Pausing in Escherichia Coli and Its Implications for Pause-mediated Transcription Regulation PDF Download
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Author: Asma Issam Hatoum
Publisher:
ISBN:
Category :
Languages : en
Pages : 302
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Author: Asma Issam Hatoum
Publisher:
ISBN:
Category :
Languages : en
Pages : 302
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Author: Brian Z. Ring
Publisher:
ISBN:
Category :
Languages : en
Pages : 292
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Author: Nicholas James Fuda
Publisher:
ISBN:
Category :
Languages : en
Pages : 202
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Most gene expression is regulated at the level of transcription, and the transition from initiation to productive elongation is a key point of regulation. This transition is accompanied by pausing of transcriptionally engaged polymerase in the promoter-proximal region of several heat shock genes. Although this mechanism of regulation was long thought to be limited to a few genes, recent evidence has indicated that pausing is wide-spread in higher eukaryotes. Therefore, it is increasingly important to understand the mechanisms controlling the paused polymerase. I have investigated how the site of pausing on Hsp70 is specified using highresolution mapping of polymerase on reporter genes with shifted pausing site sequences. The results indicate that the downstream sequence dictates pause position and the overall level of pausing. I have also used RNAi knock-down in Drosophila cell culture to study the roles of several factors in establishing, maintaining, and releasing the paused polymerase. These experiments have shown GAGA factor is required for pausing on many of its target genes, and the knock-down effects indicate it is involved in establishing the pause. In contrast, Spt5, a protein previously shown to enhance pausing in vitro, reduces pausing genome-wide by increasing levels of elongating polymerase. Two kinases, P-TEFb and CDK12, function in productive elongation. Previously our lab showed that P-TEFb inhibition prevented the transition into elongation, limiting the polymerase to the 5' end of the heat shock-induced Hsp70 gene. I mapped these polymerases in high resolution to show they occupied sites further downstream than the normal pause sites, suggesting P-TEFb activity may not solely release the paused polymerase. I also determined the localization of CDK12 on active genes. Its localization downstream of P-TEFb suggests that these kinases may have distinct functions. Finally, I have examined the role of Fcp1 in Hsp70 transcription. Our lab previously showed the CTD phosphatase Fcp1 was required for optimum expression of Hsp70 mRNA. Fcp1 knock-down reduced the heat shock levels of Pol II and increased phosphorylation of nonchromatin bound Pol II, indicating that Fcp1 recycling of RNA polymerase II to an initiationcompetent form is required for optimal Hsp70 heat shock transcription.
Author: Jeremy Gilbert Bird
Publisher:
ISBN:
Category :
Languages : en
Pages : 302
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Transcription is not necessarily uniform in rate, but instead consists of multiple periods of continuous elongation by RNA polymerase (RNAP) interrupted by occasional pausing events. These pauses have several determined origins, including the interaction of protein co-factors, the influence of transcribed RNA secondary structure, and a direct effect of the sequence composition of the DNA being transcribed. Promoter proximal pauses, in which RNAP pauses tens of nucleotides downstream of the transcription start site, have been observed in systems ranging from bacteria to more complex eukaryotes such as D. melanogaster and humans, and play important regulatory roles in organisms of all levels. In E. coli, [sigma]70-dependent promoter proximal pauses are of particular interest because lambdoid phage late gene promoters require these pauses for the loading of the anti-terminator Q protein, which is necessary for transcription of the phage late genes during lytic growth. The primary interaction that induces [sigma]70-dependent pauses is between the non-template strand of the -10-like sequence and [sigma]70 region 2. However, recent evidence identifies other important sequences including several nucleotides around the pause site itself that are required for pausing (Perdue and Roberts 2010). Mutational studies of lambdoid phage 82 promoter pR' show that a 3-bp GC-rich sequence primarily promotes pausing through the template DNA strand, but also acts through a non-template DNA strand interaction with [sigma]70. I show that this template strand sequence determines where the pause occurs. It is likely that in [sigma]-dependent promoterproximal pausing, the [sigma]70/DNA interaction anchors the elongation complex upstream of the pause site, requiring it to "scrunch" (by analogy to scrunching during initial transcription (Kapanidis et al. 2006)) and to create an enlarged DNA bubble as the active center reaches the pause site. "Scrunched" complexes are energetically strained, but stable at the pause site; my data suggest that the G/C-rich template sequence is a critical element in stabilizing the paused, scrunched structure. Mutational studies of the A/T-rich region demonstrate that the terminal nucleotide of the pause site plays an important role in pause formation, and that the base composition of the A/T-rich sequence determines the likelihood that a transcription complex escapes the pause. My data also suggest that a [sigma]70-dependent paused transcription complex enters the paused state through a pretranslocated state of the enzyme. Through these studies, I have expanded the current understanding of the protein/nucleotide and nucleotide/nucleotide interactions that constitute the [sigma]70dependent promoter proximal paused complex.
Author:
Publisher:
ISBN:
Category : Medicine
Languages : en
Pages : 2388
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Vols. for 1963- include as pt. 2 of the Jan. issue: Medical subject headings.
Author: Torben Heick Jensen
Publisher: Springer Science & Business Media
ISBN: 1441978410
Category : Medical
Languages : en
Pages : 161
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Book Description
The diversity of RNAs inside living cells is amazing. We have known of the more “classic” RNA species: mRNA, tRNA, rRNA, snRNA and snoRNA for some time now, but in a steady stream new types of molecules are being described as it is becoming clear that most of the genomic information of cells ends up in RNA. To deal with the enormous load of resulting RNA processing and degradation reactions, cells need adequate and efficient molecular machines. The RNA exosome is arising as a major facilitator to this effect. Structural and functional data gathered over the last decade have illustrated the biochemical importance of this multimeric complex and its many co-factors, revealing its enormous regulatory power. By gathering some of the most prominent researchers in the exosome field, it is the aim of this volume to introduce this fascinating protein complex as well as to give a timely and rich account of its many functions. The exosome was discovered more than a decade ago by Phil Mitchell and David Tollervey by its ability to trim the 3’end of yeast, S. cerevisiae, 5. 8S rRNA. In a historic account they laid out the events surrounding this identification and the subsequent birth of the research field. In the chapter by Kurt Januszyk and Christopher Lima the structural organization of eukaryotic exosomes and their evolutionary counterparts in bacteria and archaea are discussed in large part through presentation of structures.
Author: Gabriel Waksman
Publisher: Amer Society for Microbiology
ISBN: 9781555813017
Category : Science
Languages : en
Pages : 273
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Book Description
Structural Biology of Bacterial Pathogenesis explores recent developments in the understanding of the molecular basis of bacterial infectious diseases, from structures involved in adhesion and host recognition to those describing elements of bacterial secretion systems Compiles engaging, convenient reviews of current research Presents high–quality illustrations of key importance to structural biology Reviews comprehensively the molecular basis of bacterial pathogenesis Provides a link between pathology and its underlying molecular mechanisms Contributors are leading scientists in the fields of structural biology and bacterial infectious diseases
Author: Gregory T. Booth
Publisher:
ISBN:
Category :
Languages : en
Pages : 404
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Promoter-proximal pausing of RNA Polymerase II (Pol II) is now recognized as a ubiquitous mechanism for regulating gene expression in metazoans. By capturing engaged Pol II shortly after transcription initiation, genes are primed for activation of RNA synthesis, enabling cells to rapidly alter global transcription programs. However, despite conservation of many factors involved in establishing this regulatory platform, many eukaryotes do not control gene expression through this process. Here, the examination of the global transcriptional landscape in two distantly related yeast revealed unprecedented divergence in Pol II distributions across genes. Previously undescribed pause-like profiles were identified within promoter-proximal regions of the fission yeast, Schizosaccharomyces pombe, that are sensitive to loss of the conserved elongation factor, Spt4. Thus, fission yeast might employ a variant of the system of regulation found in higher eukaryotes In flies and mammals, Pol II arrested within the promoter proximal region of a gene can only be released through the activity of a positive-transcription elongation factor (P-TEFb), composed of kinase (Cdk9) and cyclin (CycT1/2) subunits. Investigating the functional impact of Cdk9 on transcription in fission yeast revealed that, unlike most metazoan systems, Pol II in S. pombe is capable of overcoming the early elongation barrier after kinase inhibition, although not without consequence. However, fission yeast lack the metazoan-specific negative elongation factor complex (NELF) involved in pausing, perhaps limiting their ability to control the release of Pol II through phosphorylation of the elongation complex. Ultimately, by depleting pausing factors from cell lines derived from Drosophila melanogaster, it was tested whether NELF is required for P-TEFb-regulated pause escape. While global transcription is largely unaffected by the loss of NELF, upon inhibition of Cdk9, a significant amount of Pol II is aberrantly released from the pause, suggesting reduced control of this regulation. These findings suggest that NELF may have evolutionarily refined an ancestral promoter-proximal architecture of the transcription elongation complex, giving rise to a novel mechanism for gene regulation.
Author:
Publisher:
ISBN:
Category : Medicine
Languages : en
Pages : 1456
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Author: Eric John Strobel
Publisher:
ISBN:
Category :
Languages : en
Pages : 115
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The movement of RNA polymerase (RNAP) along a DNA template is punctuated by frequent pauses during which the elongation complex persists at a single position for an extended period of time. Such pausing events have been shown to function in the regulation of gene expression at the level of transcription. A prototypical example of regulatory pausing is provided by the lambdoid phage late gene operon, in which a DNA-encoded [sigma]70-dependent pause element halts transcription to facilitate incorporation of the antiterminator Q into the paused elongation complex. Upon binding, Q triggers rapid escape of the now terminator-resistant elongation complex from the pause site. The [sigma]70-dependent pause element consists of a hexameric "-10-like sequence", which functions by capturing RNAP through contacts with the [sigma]70 subunit, and a "translocation pause element" (TPE), a pause-inducing element that underlies transcription pausing in E. coli. Here, we show that the TPE contributes to [sigma]70-dependent pausing by limiting the rate at which paused complexes attempt to escape the pause and we identify a novel sub-element that contributes to TPE function. Furthermore, we use a functional analog to show that Q drives escape from the [sigma]70-dependent pause in a process that is mechanistically related to escape from a promoter during transcription initiation.
