RNA POLYMERASE-II PROMOTER STRENGTH IN-VITRO MAY BE REDUCED BY DEFECTS AT INITIATION OR PROMOTER CLEARANCE

We have measured the ability of three TATA box promoters, adenovirus 2 major late (Ad 2 ML), Ad 2 ML with a point mutation in the TATA box, and mouse P-globin, to support abortive and productive RNA synthesis i n vitro. We have also measured the ability of these promoters to direc t the assembly of preinitiation complexes using a nuclease protection assay. The relative strengths in productive transcription, determined from the synthesis of RNAs 10 nucleotides or longer, were 12:6:1 for A d 2 ML, mouse beta-globin, and the TATA mutant of Ad 2 ML. However, th e TATA mutant was reduced only 4-fold in its ability to assemble prein itiation complexes, compared to Ad 2 ML. Complexes formed on the Ad 2 ML TATA mutant may therefore also be reduced in their ability to initi ate transcription. The mouse beta-globin promoter directed assembly an d initiation as well as Ad 2 ML, but the beta-globin transcription com plexes were less able to clear the promoter, resulting in an increase in aborted transcripts at the expense of productive RNA synthesis. We have thus shown that the transcriptional strength of eukaryotic promot ers may be determined not only at the step of transcription complex as sembly but also at the level of promoter clearance and possibly at tra nscription initiation as well.