Analysis of Nucleosome Transcription Using Single-Particle FRET

Many biological reactions including transcription of a gene are too complex and heterogeneous to be understood by studying ensembles of interacting molecules. In these cases analysis of single complexes can clarify structural and dynamic aspects of these processes. Here we report that single-particle Forster resonance energy transfer (spFRET ) microscopy is applicable to investigation of transcription through nucleosomes by an RNA polymerase . Mononucleosomes that support transcription were assembled from core histones and short DNA containing the T7A1 promoter and strong 603 nucleosome-positioning sequence. Fluorophores (Cy3 and Cy5) were introduced in the neighboring coils of nucleosome DNA in spatially close positions without disturbance of nucleosomal structure or transcription. Such labeling allows the changes in the Cy3–Cy5 distance caused by DNA uncoiling from the octamer or DNA looping to be monitored as changes in FRET efficiency. spFRET measurements for freely diffusing single nucleosomes were conducted using a laser scanning confocal microscope equipped with avalanche photodiodes. Nucleosome subpopulations that differ in FRET efficiency (i.e. in nucleosome structure) were revealed. RNA polymerase was stalled in distinct positions on the nucleosomal DNA during transcription, and the structures of these complexes were characterized with spFRET microscopy.