Abstract
Nucleosomes are fundamental repeating subunits of eukaryotic chromatin which help to package DNA tightly into the cell nucleus. During transcription, RNA polymerase (RNAP) must be able to transcribe the DNA associated with nucleosomes, yet nucleosomes are known to be major barriers to transcription. Here we use optical trapping techniques in combination with biochemical methods to study the mechanisms by which E.coli RNAP transcribes through nucleosomes. Although E. coli RNAP never encounters chromatin in vivo, its core enzyme is evolutionarily conserved and shares homology in sequence, structure and function with eukaryotic Pol II, suggesting that E. coli RNAP may be a simple system to study transcription through nucleosomes. We have constructed DNA templates, each containing a T7A1 promoter followed by a well-positioned mononucleosome. We use optical trapping to detect with high precision the position and structure of proteins before and after transcription. Our results show that RNA polymerase may backtrack when it encounters a nucleosome. Prevention of backtracking alleviates the nucleosomal barrier, promoting more efficient transcription through nucleosomes.
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