The sequence at specific positions in the early transcribed region sets the rate of transcript synthesis by RNA polymerase II in vitro.
To further understand the mechanism of promoter escape by RNA polymerase II, we have systematically investigated the effect of core promoter sequence on the rate of transcript synthesis in vitro. Chimeric and mutant promoters were made by swapping sequences between the human interleukin-2 promoter and the adenovirus major late promoter, which exhibit different rates of transcript synthesis. Kinetic studies at these promoters revealed that sequences downstream of the start sites set the rate of transcript synthesis. Specifically, the sequences at +2 and +7/+8 are critical for determining the rate; when either +2 is a C (nontemplate strand) or +7/+8 is a TT (nontemplate strand), transcript synthesis is slow. At +7/+8, the thermodynamic stability of the RNA:DNA hybrid controls the overall rate of transcript synthesis. Our data support a model in which the rate-limiting step during transcript synthesis by RNA polymerase II in vitro occurs at the point in the reaction at which early ternary complexes transform into elongation complexes.
Weaver
Jessica R
JR
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Kugel
Jennifer F
JF
Goodrich
James A
JA
D000256:Adenoviridae / Q000235:genetics; D001483:Base Sequence; D006801:Humans; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000235:genetics; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics; D013997:Time Factors; D014158:Transcription, Genetic
D007376:Interleukin-2; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012319:RNA Polymerase II