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Molecular and Cellular Biology, October 2001, p. 6429-6439, Vol. 21, No. 19
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.19.6429-6439.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

A Novel Upstream RNA Polymerase III Promoter Element Becomes Essential When the Chromatin Structure of the Yeast U6 RNA Gene Is Altered

Michael P. Martin, Valerie L. Gerlach,dagger and David A. Brow*

Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706-1532

Received 28 March 2001/Returned for modification 27 April 2001/Accepted 9 July 2001

The Saccharomyces cerevisiae U6 RNA gene, SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription of SNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position -30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele of SNR6 with decreased spacing between the A and B blocks, snr6-Delta 42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Delta 42 is much more sensitive to mutations in a (dT-dA)7 tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Delta 42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription of SNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)7 tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.

* Corresponding author. Mailing address: Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706-1532. Phone: (608) 262-1475. Fax: (608) 262-5253. E-mail: dabrow{at}facstaff.wisc.edu.

dagger Present address: CuraGen Corporation, Branford, CT 06405.

Molecular and Cellular Biology, October 2001, p. 6429-6439, Vol. 21, No. 19
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.19.6429-6439.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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