TGA1a, a tobacco DNA-binding protein, increases the rate of preinitiation complex formation in a plant in vitro transcription system [corrected

Abstract

We describe here a plant in vitro transcription system for class II promoters using wheat germ extract. In this system transcription is stimulated by TGA1a, a tobacco DNA-binding protein, and the stimulation is dependent on the presence of its cognate binding site upstream of the TATA box. Titration experiments showed that transcription initiation is more sensitive than transcription elongation to low concentrations of sarkosyl (N-lauroylsarcosine). At 0.07% sarkosyl, the formation of initiated complex is inhibited but transcription elongation is not. Under these conditions, events associated with a single round of transcription can be studied. We demonstrate that the time required for completing transcription of a 380-base-pair template is about 10 min. Addition of TGA1a increases the number of preinitiation complexes by approximately 3-fold with no significant effect on the frequency of transcription initiation from a single complex or on the rate of RNA elongation. We anticipate that this in vitro system will be valuable for the elucidation of mechanisms that regulate transcription in plants.