Oligonucleosomes as a model system for chromatin transcription: Transcription with Escherichia coli DNA-dependent RNA polymerase

Abstract

The transcription of eukaryotic genes that are organized into nucleosomal structures is likely to be influenced by the presence of the chromosomal proteins. We investigate in this paper the transcriptional behavior of series of oligonucleosomes, produced by Micrococcus nuclease digestion of nuclei, with RNA polymerase from Escherichia coli. Electrophoresis of the transcribed RNA shows that the nuclease-generated ends of such oligonucleosomes serve as artificial initiation sites, and that the templates are transcribed in full lengths. Thus the problem of inefficient initiation, which has been encountered with circular minichromosomes, can be circumvented. The influence of the nucleosomal histones on transcription was assessed by comparison with the corresponding deproteinized DNA fragments as templates. Unlike most previous reports on chromatin transcription we find for oligonucleosomes a high template activity ranging from 30 to 100% relative to their corresponding DNA fragments. The ratio of incorporation on oligonucleosomes relative to the deproteinized DNA fragments did not show a dependence on the template length, thus indicating the absence of specific effects due to long templates or to higherorder structures. From the temperature dependence of transcription it can be concluded that at 37 °C the presence of nucleosomes on the template does not affect the elongation rate. Furthermore the elongation process seems to be rate-limiting at this temperature. We propose that the lower template activity of nucleosomes compared with naked DNA can be attributed to a decreased availability of template ends as initiation sites. Stabilizing influences of the nucleosomes on the transcription process were observed: E. coli polymerase after preinitiation shows, at salt concentrations higher than 350 m m, a higher incorporation on oligonucleosomes than on DNA. Below 25 °C the nucleosomes seem to lower the activation energy for the initiation on the nuclease-generated template ends.