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J. Biol. Chem., Vol. 281, Issue 20, 14119-14128, May 19, 2006

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Quantitative Analysis of in Vivo Initiator Selection by Yeast RNA Polymerase II Supports a Scanning Model^*

From the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706

Initiation of transcription by RNA polymerase II (RNAP II) on Saccharomyces cerevisiae messenger RNA (mRNA) genes typically occurs at multiple sites 40–120 bp downstream of the TATA box. The mechanism that accommodates this extended and variable promoter architecture is unknown, but one model suggests that RNAP II forms an open promoter complex near the TATA box and then scans the template DNA strand for start sites. Unlike most protein-coding genes, small nuclear RNA gene transcription starts predominantly at a single position. We identify a highly efficient initiator element as the primary start site determinant for the yeast U4 small nuclear RNA gene, SNR14. Consistent with the scanning model, transcription of an SNR14 allele with tandemly duplicated start sites initiates primarily from the upstream site, yet the downstream site is recognized with equivalent efficiency by the diminished population of RNAP II molecules that encounter it. A quantitative in vivo assay revealed that SNR14 initiator efficiency is nearly perfect (90%), which explains the precision of U4 RNA 5' end formation. Initiator efficiency was reduced by cis-acting mutations at –8, –7, –1, and +1 and trans-acting substitutions in the TFIIB B-finger. These results expand our understanding of RNAP II initiation preferences and provide new support for the scanning model.

Received for publication, February 28, 2006 , and in revised form, March 29, 2006.

^* This work was supported by National Institutes of Health, Public Health Service Grant GM44665 (to D. A. B.). 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. The work is dedicated to the memory of Kathryn A. Kuehner (1939–2006).

¹ To whom correspondence should be addressed: Dept. of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, 1300 University Ave., Madison, WI 53706-1532. Tel.: 608-262-1475; Fax: 608-262-5253; E-mail: dabrow{at}wisc.edu.

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