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

摘要

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.