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 beta-globin, to support abortive and productive RNA synthesis in vitro. We have also measured the ability of these promoters to direct 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 Ad 2 ML, mouse beta-globin, and the TATA mutant of Ad 2 ML. However, the TATA mutant was reduced only 4-fold in its ability to assemble preinitiation complexes, compared to Ad 2 ML. Complexes formed on the Ad 2 ML TATA mutant may therefore also be reduced in their ability to initiate transcription. The mouse beta-globin promoter directed assembly and initiation as well as Ad 2 ML, but the beta-globin transcription complexes 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 promoters may be determined not only at the step of transcription complex assembly but also at the level of promoter clearance and possibly at transcription initiation as well.
Jacob
G A
GA
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
Kitzmiller
J A
JA
Luse
D S
DS
D000260:Adenoviruses, Human / Q000201:enzymology / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D015870:Gene Expression; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D016385:TATA Box; D013698:Templates, Genetic; D014158:Transcription, Genetic
D005914:Globins; D012319:RNA Polymerase II