Protein-DNA Interactions Studies with Single Tethered Molecule Techniques

Nir, Guy; Lindner, Moshe; Garini, Yuval

doi:10.5772/38416

Cited by 1 publication

(1 citation statement)

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“…Recently, Garini et al proposed to use confocal microscopy to observe the excursion of the labeled bead in three-dimensions (Nir, Lindner, & Garini, 2012). Hidden Markov models using variational Bayesian methods have also been implemented to improve resolution and objectively assign conformational states (Johnson, van de Meent, Phillips, Wiggins, & Linden, 2014).…”

Section: Introductionmentioning

confidence: 99%

Multiplexed, Tethered Particle Microscopy for Studies of DNA-Enzyme Dynamics

Ucuncuoglu

Schneider

Weeks

et al. 2017

Methods in Enzymology

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DNA is the carrier of genetic information, and as such, is at the center of most essential cellular processes. To regulate its physiological function, specific proteins and motor enzymes constantly change its conformational state with well controlled dynamics. Twenty-five years ago, Schafer, Gelles, Sheetz and Landick employed the Tethered Particle Motion (TPM) technique for the first time to study transcription by RNA polymerase at the single-molecule level. TPM has since then remained one of the simplest, most affordable and yet incisive single-molecule techniques available. It is an in vitro technique which allows investigation of DNA-protein interactions that change the effective length of a DNA tether. In this chapter, we will describe a recent strategy to multiplex TPM which substantially increases the throughput of TPM experiments, as well as a simulation to estimate the time-resolution of experiments, such as transcriptional elongation assays, in which lengthy time averaging of the signal is impossible due to continual change of the DNA tether length. These improvements allow efficient study of several DNA-protein systems, including transcriptionally active DNA-RNA polymerase I complexes and DNA-gyrase complexes.

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Section: Introductionmentioning

confidence: 99%