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J. Biol. Chem., Vol. 281, Issue 33, 23533-23544, August 18, 2006

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Mechanism of Instability in Abortive Cycling by T7 RNA Polymerase^*

From the Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003-9336

Abortive transcription, the premature release of short transcripts 2–8 bases in length, is a unique feature of transcription, accompanying the transition from initiation to elongation in all RNA polymerases. The current study focuses on major factors that relate to the stability of initially transcribing abortive complexes in T7 RNA polymerase. Building on previous studies, results reveal that collapse of the DNA from the downstream end of the bubble is a major contributor to the characteristic instability of abortive complexes. Furthermore, transcription from a novel DNA construct containing a nick between positions –14 and –13 of the nontemplate strand suggests that the more flexible promoter reduces somewhat the strain inherent in initially transcribing complexes, with a resulting decrease in abortive product release. Finally, as assessed by exonuclease III footprinting and transcription profiles, a DNA construct defective in bubble collapse specifically from the downstream end exhibits less abortive cycling and little perturbation of the final transition to elongation, including the process of promoter release.

Received for publication, April 27, 2006 , and in revised form, June 20, 2006.

^* This work was supported by National Institutes of Health Grant 1R01 GM55002 (to C. T. M.). 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 Chemistry, University of Massachusetts Amherst, 710 N. Pleasant St., LGRT 701, Amherst, MA 01003-9336. Tel.: 1-413-545-3299; Fax: 1-413-545-4490; E-mail: CMartin{at}chem.umass.edu.

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