Studying &#963;<sup>54</sup>-dependent transcription at the single-molecule level using alternating-laser excitation (ALEX) spectroscopy

M. Heilemann; K. Lymperopoulos; S. R. Wigneshweraraj; M. Buck; A. N. Kapanidis

doi:10.1117/12.729521

13 July 2007 Studying σ⁵⁴-dependent transcription at the single-molecule level using alternating-laser excitation (ALEX) spectroscopy

M. Heilemann, K. Lymperopoulos, S. R. Wigneshweraraj, M. Buck, A. N. Kapanidis

Author Affiliations +

Proceedings Volume 6633, Biophotonics 2007: Optics in Life Science; 66332K (2007) https://doi.org/10.1117/12.729521
Event: European Conferences on Biomedical Optics, 2007, Munich, Germany

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.

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M. Heilemann, K. Lymperopoulos, S. R. Wigneshweraraj, M. Buck, and A. N. Kapanidis "Studying σ⁵⁴-dependent transcription at the single-molecule level using alternating-laser excitation (ALEX) spectroscopy", Proc. SPIE 6633, Biophotonics 2007: Optics in Life Science, 66332K (13 July 2007); https://doi.org/10.1117/12.729521

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KEYWORDS

Fluorescence resonance energy transfer

Luminescence

Spectroscopy

Fluorescence spectroscopy

Proteins

Single molecule spectroscopy

Data hiding

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