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J. Biol. Chem., Vol. 280, Issue 49, 40707-40713, December 9, 2005

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Extended Upstream A-T Sequence Increases T7 Promoter Strength^*

From the Department of Biochemistry, University of Medicine and Dentistry of New Jersey (UMDNJ) Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Bacteriophage T7 promoters contain a consensus sequence from -17 to +6 relative to the transcription start site, +1. In addition, the strong class III promoters are characterized by an extended AT-rich region upstream of -17, which is often interrupted by one or more GC base pairs in the weaker class II promoters. Herein we studied the role of the AT-rich region upstream of -17 in transcription regulation of T7 RNA polymerase. Equilibrium DNA binding studies with promoter fragments of consensus sequence truncated at various positions between -17 and -27 showed that the polymerase-promoter complex is significantly stabilized as the upstream AT-rich sequence is extended to and beyond -22. Similarly, promoters in which the AT-rich region from -17 to -22 is interrupted by several GC base pairs showed weak binding. Kinetic studies indicated that the presence of extended AT-rich sequence slows the dissociation rate constant of the polymerase-promoter complex and slightly stimulates the association rate constant, thereby increasing the stability of the complex. Measurement of the transcription activity revealed that the extended AT-rich region does not affect the kinetics of abortive synthesis up to the formation of 8-nucleotide RNA but causes accumulation of longer abortive products between 9 and 13 nucleotides. The observed effects of the upstream DNA region were AT sequence-specific, and the results suggested a larger role for the extended AT-rich sequence that has been unappreciated previously. We propose that the AT-rich DNA sequence upstream of -17 plays a role in modulating the efficiency of transcription initiation by affecting both the affinity of T7 RNA polymerase for the promoter and the efficiency of promoter clearance.

Received for publication, July 22, 2005 , and in revised form, October 5, 2005.

^* This work was supported by National Institutes of Health Grant GM51966 (to S. S. P.). 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 To whom correspondence should be addressed: Dept. of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes La., Piscataway, NJ 08854. Tel.: 732-235-3372; Fax: 732-235-4783; E-mail: patelss{at}umdnj.edu.

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