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Molecular and Cellular Biology, March 2007, p. 1545-1557, Vol. 27, No. 5
0270-7306/07/$08.00+0     doi:10.1128/MCB.00773-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Requirement of Nhp6 Proteins for Transcription of a Subset of tRNA Genes and Heterochromatin Barrier Function in Saccharomyces cerevisiae{triangledown}

Priscilla Braglia,1,{dagger} Sandra L. Dugas,2 David Donze,2 and Giorgio Dieci1*

Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, 43100 Parma, Italy,1 Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 708032

Received 3 May 2006/ Returned for modification 11 August 2006/ Accepted 4 December 2006

A key event in tRNA gene (tDNA) transcription by RNA polymerase (Pol) III is the TFIIIC-dependent assembly of TFIIIB upstream of the transcription start site. Different tDNA upstream sequences bind TFIIIB with different affinities, thereby modulating tDNA transcription. We found that in the absence of Nhp6 proteins, the influence of the 5'-flanking region on tRNA gene transcription is dramatically enhanced in Saccharomyces cerevisiae. Expression of a tDNA bearing a suboptimal TFIIIB binding site, but not of a tDNA preceded by a strong TFIIIB binding region, was strongly dependent on Nhp6 in vivo. Upstream sequence-dependent stimulation of tRNA gene transcription by Nhp6 could be reproduced in vitro, and Nhp6 proteins were found associated with tRNA genes in yeast cells. We also show that both transcription and silencing barrier activity of a tDNAThr at the HMR locus are compromised in the absence of Nhp6. Our data suggest that Nhp6 proteins are important components of Pol III chromatin templates that contribute both to the robustness of tRNA gene expression and to positional effects of Pol III transcription complexes.

* Corresponding author. Mailing address: Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Viale G.P. Usberti 23A, 43100 Parma, Italy. Phone: 39-0521-905649. Fax: 39-0521-905151. E-mail: giorgio.dieci{at}unipr.it.

{triangledown} Published ahead of print on 18 December 2006.

{dagger} Present address: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

Molecular and Cellular Biology, March 2007, p. 1545-1557, Vol. 27, No. 5
0270-7306/07/$08.00+0     doi:10.1128/MCB.00773-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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