Abstract

We present singlc-molcculc fluorescence studies of σ⁵⁴-dcpcndcnt gene-transcription complexes using singlemolecule fluorescence resonance energy transfer (smFRET) and alternating-laser excitation (ALEX) spectroscopy. The ability to study one biomolcculc 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 sitc-specificallv labeled promoter-DNA fragments, we demonstrate that we can observe single diffusing σ⁵⁴-DNA and transcription-initiation RNA polymerase-σ"⁵⁴- 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.

© 2007 SPIE