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Partial purification of plant transcription factors. II. An in vitro transcription system is inefficient

Flynn, P.A., Davis, E.A., Ackerman, S.
Plant molecular biology 1987 v.9 no.2 pp. 159-169
Triticum aestivum, DNA-binding proteins, DNA-directed RNA polymerase, embryo (plant), plant extracts, gene expression, purification, inhibitors, transcription (genetics)
Crude wheat germ nuclear extracts contain many inhibitors of transcription which need to be removed before an active system can be developed. Using ion exchange column chromatography, to resolve RNA polymerase II transcription components we can identify at least four fractions required for transcription by their ability to interact with, or substitute for, particular HeLa fractions. Inhibitors can be removed by a second or third chromatographic process applied to each fraction. Two plant fractions can each effectively replace the corresponding fraction in a HeLa transcription system, and the wheat fractions can work together and replace two HeLa fractions. These plant factors chromatograph identically to HeLa factors on ion exchange columns. The third fraction does not fully substitute for the corresponding HeLa fraction, but can complement this HeLa fraction when both are added at half-optimal levels. An in vitro plant system consisting of four plant chromatographic fractions will selectively transcribe a gene, but only at very low efficiency. The apparent block to greater efficiency is in elongation of the RNA past the 20-30n size.