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Proceedings Paper

Studying σ54-dependent transcription at the single-molecule level using alternating-laser excitation (ALEX) spectroscopy
Author(s): M. Heilemann; K. Lymperopoulos; S. R. Wigneshweraraj; M. Buck; A. N. Kapanidis
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Paper Abstract

We present single-molecule fluorescence studies of σ54-dependent gene-transcription complexes using singlemolecule fluorescence resonance energy transfer (smFRET) and alternating-laser excitation (ALEX) spectroscopy. The ability to study one biomolecule at the time allowed us to resolve and analyze sample heterogeneities and extract structural information on subpopulations and transient intermediates of transcription; such information is hidden in bulk experiments. Using site-specifically labeled σ54 derivatives and site-specifically labeled promoter-DNA fragments, we demonstrate that we can observe single diffusing σ54-DNA and transcription-initiation RNA polymerase-σ54- DNA complexes, and that we can measure distances within such complexes; the identity of the complexes has been confirmed using electrophoretic-mobility-shift assays. Our studies pave the way for understanding the mechanism of abortive initiation and promoter escape in σ54-dependent transcription.

Paper Details

Date Published: 13 July 2007
PDF: 10 pages
Proc. SPIE 6633, Biophotonics 2007: Optics in Life Science, 66332K (13 July 2007); doi: 10.1117/12.729521
Show Author Affiliations
M. Heilemann, Univ. of Oxford (United Kingdom)
K. Lymperopoulos, Univ. of Oxford (United Kingdom)
S. R. Wigneshweraraj, Imperial College London (United Kingdom)
M. Buck, Imperial College London (United Kingdom)
A. N. Kapanidis, Univ. of Oxford (United Kingdom)

Published in SPIE Proceedings Vol. 6633:
Biophotonics 2007: Optics in Life Science
Jürgen Popp; Gert von Bally, Editor(s)

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