Lac repressor (LacI) is a protein representative of a family of DNA binding proteins called Transcription Factors (TFs). These proteins regulate gene expression upon binding to a specific DNA sequence, called target or cognate. LacI cognate site is called operator (O1). The association rate to the target is limited by the time required to search the short operator sequence within the whole genome. A theoretical calculation, based on random collision during 3D-diffusion, gives a rate of 108 M-1s-1. Nevertheless, the first measurements of association rate produced a value of 1010 M-1s-1, two orders of magnitude faster than the theoretical value. The most accredited model to explain this discrepancy is ‘facilitated diffusion’, according to which the protein, before encountering the target sequence, alternates between 3D-diffusion and random association to non-cognate DNA; during such non-specific interactions the protein undergoes 1D-diffusion along DNA.The time intervals during which the search dimensionality is reduced from 3D to 1D provide the basis for acceleration of target finding. Here, I measured diffusion properties of LacI varying the force applied on the extremities of the DNA molecule. This was made possible by an experimental set-up combining single-molecule nanometric localization, optical trapping and microfluidics, thus enabling to track a single, fluorescently labeled LacI molecule bound to a DNA molecule suspended in solution, far from the cover slip. The double optical tweezers employed allow application of precisely controlled tension on DNA. To localize the protein, I used a recently published rapid and accurate algorithm based on the calculation of the radial symmetry centre. From the protein trajectories, we obtained the 1D diffusion coefficient (D1D) and sliding length (the contour distance (xmax-xmin) traveled by the protein before dissociation) at different DNA tensions: 1, 10, 20 and 30 pN. I found a correlation between these two parameters, which present a parabolic dependence on DNA tension.

COMBINING SINGLE-MOLECULE LOCALIZATION, OPTICAL TRAPPING AND MICROFLUIDICS FOR THE STUDY OF PROTEIN-DNA INTERACTION / Gionata Belcastro. - (2013).

COMBINING SINGLE-MOLECULE LOCALIZATION, OPTICAL TRAPPING AND MICROFLUIDICS FOR THE STUDY OF PROTEIN-DNA INTERACTION

BELCASTRO, GIONATA
2013

Abstract

Lac repressor (LacI) is a protein representative of a family of DNA binding proteins called Transcription Factors (TFs). These proteins regulate gene expression upon binding to a specific DNA sequence, called target or cognate. LacI cognate site is called operator (O1). The association rate to the target is limited by the time required to search the short operator sequence within the whole genome. A theoretical calculation, based on random collision during 3D-diffusion, gives a rate of 108 M-1s-1. Nevertheless, the first measurements of association rate produced a value of 1010 M-1s-1, two orders of magnitude faster than the theoretical value. The most accredited model to explain this discrepancy is ‘facilitated diffusion’, according to which the protein, before encountering the target sequence, alternates between 3D-diffusion and random association to non-cognate DNA; during such non-specific interactions the protein undergoes 1D-diffusion along DNA.The time intervals during which the search dimensionality is reduced from 3D to 1D provide the basis for acceleration of target finding. Here, I measured diffusion properties of LacI varying the force applied on the extremities of the DNA molecule. This was made possible by an experimental set-up combining single-molecule nanometric localization, optical trapping and microfluidics, thus enabling to track a single, fluorescently labeled LacI molecule bound to a DNA molecule suspended in solution, far from the cover slip. The double optical tweezers employed allow application of precisely controlled tension on DNA. To localize the protein, I used a recently published rapid and accurate algorithm based on the calculation of the radial symmetry centre. From the protein trajectories, we obtained the 1D diffusion coefficient (D1D) and sliding length (the contour distance (xmax-xmin) traveled by the protein before dissociation) at different DNA tensions: 1, 10, 20 and 30 pN. I found a correlation between these two parameters, which present a parabolic dependence on DNA tension.
2013
Professor Francesco Saverio Pavone
ITALIA
Gionata Belcastro
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/816099
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