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J. Biol. Chem., Vol. 280, Issue 48, 39860-39869, December 2, 2005

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The Sequence at Specific Positions in the Early Transcribed Region Sets the Rate of Transcript Synthesis by RNA Polymerase II in Vitro^*

From the Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215

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.

Received for publication, August 24, 2005 , and in revised form, October 3, 2005.

^* This work was supported in part by United States Public Health Service Grant GM55235 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by National Institutes of Health Predoctoral Training Grant T32 GM07135.

² To whom correspondence may be addressed. Tel.: 303-735-0955; Fax: 303-492-5894; E-mail: jennifer.kugel{at}colorado.edu. ³ To whom correspondence may be addressed. Tel.: 303-492-3273; Fax: 303-492-5894; E-mail: james.goodrich{at}colorado.edu.

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