Han, Lin (2008) In vitro DNA mechanics in gene regulation : one molecule at a time. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-01282008-150852
The biological significance of DNA is primarily attributed to its sequence information. On the other hand, the mechanical properties of DNA can play a critical role in a wide variety of biological processes. One prime example is DNA looping in the context of transcriptional regulation. The emergence of single molecule tracking techniques in the last two decades presents an unprecedented opportunity for studying looping kinetics. One such powerful technique, tethered particle motion (TPM), harnesses the Brownian motion of a microsphere as a means of reporting on the excursion of its tethered molecule, such as DNA. The present work focuses on a looping system found in Escherichia coli, which is mediated by the Lac repressor (LacI) protein. TPM is used to measure individual, real-time looping/unlooping events in DNA of various length and sequence characteristics. By monitoring the magnitude, frequency, and time interval of these features while tuning different parameters, such as LacI concentration, DNA length and DNA sequence, one can survey a host of important information about looping kinetics. A measurement of the LacI concentration dependence of looping probability was found to be in quantitative agreement with a simple thermodynamic model, which also led to the measurement of free energy of LacI-mediated looping, the first such measurement in a single molecule, in vitro setting. A quantitative characterization of free energy was obtained under conditions of different inter-operator spacing, systematically varied from 300 to 310 base pairs in one-base-pair increments. An important conclusion from this study is that free energy is modulated by DNA’s helical structure, yet the energy difference between the aligned and unaligned operator configurations is small compared to expectation from simple polymer physics. TPM measurements also revealed an additional looped state, lending support to the hypothesis that two distinct conformations of LacI, the closed and open forms, can coexist. This study also confirmed that the presence of certain DNA sequences, particularly TA pairs in the minor groove of the nucleosomal positioning sequence, makes DNA substantially softer than a corresponding random sequence. This provides direct support for the notion of sequence-dependent DNA elasticity. Finally, a surprising result is that loops as short as 100 base pairs-only two-thirds the persistence length of DNA-can form by LacI-DNA binding. Classical elasticity theory almost forbids this, suggesting that LacI itself plays a more direct role in the bending process, or classical understanding of DNA elasticity breaks down at length scales comparable to its persistence length.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||DNA; Gene Regulation; Looping; Tethered Particle Motion|
|Degree Grantor:||California Institute of Technology|
|Division:||Engineering and Applied Science|
|Major Option:||Applied Physics|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||10 July 2007|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||21 Feb 2008|
|Last Modified:||26 Dec 2012 02:29|
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