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Abstract
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.
We thank Reid Johnson (UCLA) for anti-Nhp6 antiserum; Michael Snyder (Yale University), Marie-Claude Marsolier (CEA-Saclay), and Tiziana Lodi (University of Parma) for strains and plasmids; Joël Acker (CEA-Saclay) for pure, recombinant Bdp1 protein; and Mircko Alzapiedi, Milena Preti, Ilaria Lamberto, and Gloria Fiorino (University of Parma) for help and discussions. Special thanks to Nick Proudfoot (University of Oxford) for allowing some experiments to be carried out in his laboratory.
This work was supported by the Human Frontier Science Program Organization (grant RGY0011/2002-C to G.D. and D.D.); by the Italian Ministry of Education, University and Research (MIUR, 2005 PRIN and FIRB Programs); and by the National Science Foundation (grant MCB-0342113 to D.D.).