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Genome-Wide Effects of ppGpp Binding to RNA Polymerase on E. coli Gene Expression
Başlık:
Genome-Wide Effects of ppGpp Binding to RNA Polymerase on E. coli Gene Expression
Yazar:
Sánchez-Vázquez, Patricia, author.
ISBN:
9780438056435
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (326 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: Richard L. Gourse Committee members: Michael M. Cox; Robert Landick; Jue D. Wang; Karen M. Wassarman; James C. Weisshaar.
Özet:
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.
Notlar:
School code: 0262
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(682895.1) | 682895-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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