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J. Biol. Chem., Vol. 281, Issue 33, 23945-23957, August 18, 2006

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A Minimal Promoter for TFIIIC-dependent in Vitro Transcription of snoRNA and tRNA Genes by RNA Polymerase III^*

Elisa Guffanti¹, Roberto Ferrari¹, Milena Preti, Matteo Forloni, Olivier Harismendy, Olivier Lefebvre, and Giorgio Dieci²

From the Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, 43100 Parma, Italy and the Service de Biochimie et Génétique Moléculaire, Bâtiment 144, CEA/Saclay, 91191 Gif-sur-Yvette Cedex, France

The Saccharomyces cerevisiae SNR52 gene is unique among the snoRNA coding genes in being transcribed by RNA polymerase III. The primary transcript of SNR52 is a 250-nucleotide precursor RNA from which a long leader sequence is cleaved to generate the mature snR52 RNA. We found that the box A and box B sequence elements in the leader region are both required for the in vivo accumulation of the snoRNA. As expected box B, but not box A, was absolutely required for stable TFIIIC, yet in vitro. Surprisingly, however, the box B was found to be largely dispensable for in vitro transcription of SNR52, whereas the box A-mutated template effectively recruited TFIIIB; yet it was transcriptionally inactive. Even in the complete absence of box B and both upstream TATA-like and T-rich elements, the box A still directed efficient, TFIIIC-dependent transcription. Box B-independent transcription was also observed for two members of the tRNA^Asn(GTT) gene family, but not for two tRNA^Pro(AGG) gene copies. Fully recombinant TFIIIC supported box B-independent transcription of both SNR52 and tRNA^Asn genes, but only in the presence of TFIIIB reconstituted with a crude B'' fraction. Non-TFIIIB component(s) in this fraction were also required for transcription of wild-type SNR52. Transcription of the box B-less tRNA^Asn genes was strongly influenced by their 5'-flanking regions, and it was stimulated by TBP and Brf1 proteins synergistically. The box A can thus be viewed as a core TFIIIC-interacting element that, assisted by upstream TFIIIB-DNA contacts, is sufficient to promote class III gene transcription.

Received for publication, December 27, 2005 , and in revised form, May 31, 2006.

^* This work was supported by the Human Frontier Science Program Organization (Grant RGY0011/2002-C, to G. D.) and by the Italian Ministry of Education, University and Research (FIRB and COFIN Programs). 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 Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Parco Area delle Scienze 23/A, 43100 Parma, Italy. Tel.: 39-0521-905649; Fax: 39-0521-905151; E-mail: giorgio.dieci{at}unipr.it.

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