Selective and accurate initiation of transcription at the T-DNA promoter in a soluble chromatin extract from wheat germ

Abstract

An in vitro assay system was developed for accurate transcription of the octopine type T-DNA gene in a wheat germ extract. The system consists of the protein fraction extracted from the chromatin of wheat germ, substrates and exogenously added DNA. Specific initiation at the promoter was determined by a combination of primer extension analysis and size analysis of the transcripts synthesized from DNA templates of various molecular sizes. Synthesis of the transcripts was sensitive to alpha-amanitin. With truncated DNA templates containing the intact promoter and the proximal transcribing region of several hundred base pairs of the T-DNA, run-off transcripts of the expected size originating at the authentic promoter were synthesized together with relatively small amounts of prematurely terminated RNA molecules. On fractionation of the chromatin protein extract by DEAE-cellulose column chromatography, the fraction eluted with 0.3 M KCl showed no activity by itself for specific initiation of transcription at the promoter by RNA polymerase II. The activity was however restored by adding the fraction eluted with 0.15 M KCl, and the reconstituted RNA polymerase fraction correctly initiated transcription at the authentic promoter.