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J. Biol. Chem., Vol. 280, Issue 20, 19551-19562, May 20, 2005

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Sequence Context Effects on Oligo(dT) Termination Signal Recognition by Saccharomyces cerevisiae RNA Polymerase III^*

Priscilla Braglia, Riccardo Percudani, and Giorgio Dieci

From the Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Parco Area delle Scienze 23/A, 43100 Parma, Italy

Eukaryotic RNA polymerase (Pol) III terminates transcription at short runs of T residues in the coding DNA strand. By genomic analysis, we found that T₅ and T₄ are the shortest Pol III termination signals in yeasts and mammals, respectively, and that, at variance with yeast, oligo(dT) terminators longer than T₅ are very rare in mammals. In Saccharomyces cerevisiae, the strength of T₅ as a terminator was found to be largely influenced by both the upstream and the downstream sequence context. In particular, the CT sequence, which is naturally present downstream of T₅ in the 3'-flank of some tDNAs, was found to act as a terminator-weakening element that facilitates translocation by reducing Pol III pausing at T₅. In contrast, tDNA transcription termination was highly efficient when T₅ was followed by an A or G residue. Surprisingly, however, when a termination-proficient T₅ signal was taken out from the tDNA context and placed downstream of a fragment of the SCR1 gene, its termination activity was compromised, both in vitro and in vivo. Even the T₆ sequence, acting as a strong terminator in tRNA gene contexts, was unexpectedly weak within the SNR52 transcription unit, where it naturally occurs. The observed sequence context effects reflect intrinsic recognition properties of Pol III, because they were still observed in a simplified in vitro transcription system only consisting of purified RNA polymerase and template DNA. Our findings strengthen the notion that termination signal recognition by Pol III is influenced in a complex way by the region surrounding the T cluster and suggest that read-through transcription beyond T clusters might play a significant role in the biogenesis of class III gene products.

Received for publication, October 28, 2004 , and in revised form, March 18, 2005.

^* This study was supported by Human Frontier Science Program Organization Grant RGY0011/2002-C and by the Italian Ministry of Education, University, and Research (MIUR, 2003 COFIN Program). 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.

To whom correspondence should be addressed. Tel.: 39-0521-905649; Fax: 39-0521-905151; E-mail: giorgio.dieci{at}unipr.it.

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