Initiator-dependent transcription in vitro by a wheat germ chromatin extract

Abstract

The development of plant in vitro transcription systems transcribing faithfully and efficiently from a broad range of plant nuclear promoters has remained a challenge. We examined the nucleotide sequence requirements for faithful and efficient transcription in a wheat germ chromatin extract (Yamazaki et al., Plant Mol Biol Rep 8: 114-123). The wheat germ chromatin extract was tested with a series of chimeric promoter constructs containing plant promoter sequences upstream from the TATA box, TATA boxes, and cap-site sequences (from -10 to +14, relative to the major in vivo initiation site) in different combinations. The plant extract transcribed faithfully from several chimeric promoters containing the capsite sequence of the parsley chalcone synthase promoter. The transcription was sensitive to the RNA polymerase II-specific inhibitor alpha-amanitin and was only dependent on the chalcone synthase cap-site sequence which therefore fulfils the operational criteria for a plant initiator element. Mutations of the putative chalcone synthase initiator element defined a core sequence '5'TAACAAC' around the initiation site that was necessary for efficient transcription in vitro. In contrast to the extract, purified wheat germ RNA polymerase II showed no preference for transcription from the major chalcone synthase in vivo initiation site.

Enhancers and core promoter elements are essential for the activity of a cryptic gene activation sequence from tobacco, tCUP

“…Most eukaryotic gene promoters contain a TATA element, and mutations in this sequence result in severely reduced transcription eciency in vivo and in vitro (Zhu et al 1995;Yamaguchi et al 1998). A second type of core promoter element, called an initiator (Inr), has also been found to anchor the preinitiation complex (Javahery et al 1994;Schweizer and Mosinger 1994;Zhu et al 1995;Smale 1997). In the absence of a TATA box, Inr can facilitate the initiation of transcription (Weis and Reinberg 1997).…”

Plant in Vitro Transcription Systems

“…One way to enhance the detection of this procedure is the use of poly (dA) tail at the end of the template and subsequent selection of de novo full-length in vitro transcripts using oligo (dT) columns (30). Lower sensitivities are possible with this method because of the limitation of using linear templates, which are less active than circular templates (33,48). Limited success in the past might result from the use of linear DNA templates in this assay.…”

“…Wheat germ is essentially a dormant material and may be deficient or inactivated with regard to factors required for general transcription (30). It is known to contain several inhibitors of the transcription that need to be removed before active systems can be developed (16,18,33). Extracts of isolated wheat shoot nuclei also contain inhibitors for transcription in a HeLa cell extract (20).…”

“…In their analysis, requirement for circular DNA was obligatory, and presence of 2 mM Mg 2+ was preferred over 1 mM Mn 2+ . Moreover, the in vitro transcription initiation site of the parsley pathogenesis-related protein 2 promoter was different from the in vivo site [24 nt upstream from the in vivo site (33)]. These observations suggested that the wheat germ chromatin extract represented only a partly functional in vitro system, and in vitro systems from wheat germ would need further refinement before they could be used for transcription analysis using genuine plant promoters.…”

In VitroTranscription in the Presence of DNA Oligonucleotides Can Generate Strong Anomalous Initiation Sites

“…In addition to HeLa nuclear extracts, of single-stranded oligonucleotide (õ2-fold molar exextracts isolated from Drosophila (4, 5), Xenopus laevis cess relative to template). We surmise that a comple- (1,9), wheat germ (16), and yeast (17) have also been mentary interaction between the single-stranded oliemployed for this commonly used assay.…”