In vitro transcription through nucleosomes by T7 RNA polymerase

Abstract

The present work examines the fate of nucleosomes after in vitro transcription of a 1400 bp DNA template containing the mouse alpha-globin sequences and the promoter of T7 RNA polymerase. Naked and nucleosome-bearing templates (containing about four or seven histone H1-lacking particles per template) have been studied by sedimentation, gel electrophoresis, digestion with restriction nucleases and electron microscopy. Both naked and nucleosome-organized templates could be transcribed in vitro by the T7 polymerase. With all types of templates, both full length and shorter transcripts were obtained. The incomplete transcripts were represented by many distinct bands, pointing to the presence of multiple stops in the process of elongation. The electrophoretic pattern of the transcripts was identical in naked and in nucleosome-containing templates, showing that the stops depended on some particular DNA sequences and not on the presence of nucleosomes. The efficiency of transcription in the presence of nucleosomes was decreased owing to three different factors: (i) blocked initiation in a fraction of the templates which had their promoters occupied by a nucleosome; (ii) a decreased rate of elongation and (iii) a lag period of initiation. Sedimentation velocity, electrophoretic mobility and protection of four different restriction sites of the templates demonstrated that T7 polymerase transcribed through nucleosomes without their displacement.