RNA polymerase II transcription complex assembly in nuclear extracts

In vitro transcription systems based on nuclear extracts of eukaryotic cells continue to be valuable experimental systems for assessing function of promoter sequences and defining new activities involved in transcription complex assembly and activity, but many aspects of such systems have not been experimentally examined. Here, transcription complex assembly on the promoter from the long terminal repeat of mouse mammary tumor virus was assessed in vitro with a transcription system derived from nuclear extracts of cultured HeLa cells. The extent of preinitiation complex assembly on the promoter was limited by the availability of template, even though only a small fraction of the template present in the assays participated in transcription. These results support a model for transcription complex assembly in which template DNA has two alternative fates, one leading to assembly of a functional transcription complex, and another that leads to irreversible template inactivation. The observed kinetics of assembly reflects loss of template by both pathways and is dominated by a relatively rapid rate of template inactivation. Supplementing nuclear extracts with purified TATA binding protein increased the extent as well as the apparent rate of assembly. Both effects can be explained by a TATA binding protein-dependent increase in the rate of assembly that leads to altered partitioning of template between competing pathways.