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Assessing the Kinetic Mechanism(s) by Which Secondary Channel Binding Factors in Escherichia coli Regulate Transcription Initiation
Başlık:
Assessing the Kinetic Mechanism(s) by Which Secondary Channel Binding Factors in Escherichia coli Regulate Transcription Initiation
Yazar:
Stumper, Sarah, author.
ISBN:
9780438051782
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (117 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: Jeff Gelles Committee members: Steve Buratowski; Susan Lovett; Chris Miller.
Özet:
The synthesis of RNA molecules from a DNA template, transcription, is a highly conserved process essential to cellular survival. Due to its importance, the transcription cycle is subject to strict regulation. RNA polymerase (RNAP) is the molecular machine responsible for transcription and thus a target for regulation by accessory proteins called transcription factors. To better understand the mechanisms by which these proteins modulate the catalytic properties of RNAP, it is essential to know the dynamics of their binding to and release from RNAP. I investigated in vitro how two proteins that bind within the highly conserved secondary channel of Escherichia coli RNAP, GreB and DksA, function in the regulation of transcription initiation. I used single molecule fluorescence microscopy to directly observe and quantitatively define the kinetic mechanisms involved in regulation of initiation by GreB and DksA. Using GreB as a model secondary channel binding factor to investigate the inhibitory function of these factors, we showed that inhibition occurs via a "delayed inhibition" mechanism in which the transcription factor loads onto free RNAP and only later blocks initiation. Additionally, we sought to define how these proteins compete with one another for binding to free RNAP to perform their regulatory duties and found that GreB and DksA bind distinct subpopulations of free RNAP and that the small molecule ppGpp can shift RNAP preference in favor of DksA binding and away from GreB binding. Finally, we detailed the mechanism by which DksA and ppGpp stimulate initiation at an amino acid biosynthesis promoter; binding of DksA and ppGpp to free RNAP increase the rate of RNAP binding to the promoter, thus increasing the initiation rate. Taken together, these results provide insight as to how secondary channel binding proteins operate within the cell to regulate transcription initiation in response to various environmental cues.
Notlar:
School code: 0021
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
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XX(679877.1) | 679877-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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