Transcription of chromatin in vitro

Abstract

The interaction of Escherichia coli RNA polymerase with calf thymus DNA and chromatin has been studied under conditions which permit direct measurement of binding sites capable of supporting chain initiation. The number of such sites was determined by counting the number of growing RNA chains, using either sucrose gradient ultracentrifugation or incorporation of [γ- 32P]ATP and [γ- 32P]GTP. The number of sites was also determined by titrating template with polymerase and counting the number of molecules bound at the end-point. Both measurements show that there is one site per 1000 to 1400 nucleotide pairs on DNA and one tenth that number on chromatin. The rate of chain elongation on chromatin is about one-third that on DNA, under the assay conditions used. Measurements were made under conditions in which chromatin was soluble. The results show that the number of binding sites for polymerase on chromatin is much smaller than on DNA, but that all bound enzyme molecules are capable of chain elongation. They also show that chromatin solubility is not a factor in template restriction. Studies of chromatin template activity in vitro have generally made use of the rate of RNA synthesis as a measure of the amount of template present. The methods presented here make it possible to distinguish between the number of growing chains and the rate of chain elongation and therefore provide an unambiguous determination of template concentration.