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Nucleic Acids Research Advance Access published online on July 10, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm517
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Zinc fingers 1 and 7 of yeast TFIIIA are essential for assembly of a functional transcription complex on the 5 S RNA gene

Karen Rothfels1, Owen Rowland1 and Jacqueline Segall1,2,*

1Department of Biochemistry and 2Department of Molecular and Medical Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8

*To whom correspondence should be addressed. Tel: 1 416 978 4981; Fax: 1 416 978 8548; Email: j.segall{at}utoronto.ca

Received May 6, 2007. Revised June 11, 2007. Accepted June 15, 2007.

The binding of transcription factor (TF) IIIA to the internal control region of the 5 S RNA gene is the first step in the assembly of a DNA–TFIIIA–TFIIIC– TFIIIB transcription complex, which promotes accurate transcription by RNA polymerase III. With the use of mutations that are predicted to disrupt the folding of a zinc finger, we have examined the roles of zinc fingers 1 through 7 of yeast TFIIIA in the establishment of a functional transcription complex both in vitro and in vivo. Our data indicate that, in addition to their role in DNA binding, the first and seventh zinc fingers contribute other essential roles in the assembly of an active transcription complex. Alanine-scanning mutagenesis identified residues within zinc finger 1 that are not required for DNA binding but are required for incorporation of TFIIIC into the TFIIIA–DNA complex. Although disruption of zinc finger 2 or 3 had a deleterious effect on the activity of TFIIIA both in vitro and in vivo, we found that increasing the level of their in vivo expression allowed these mutant proteins to support cell viability. Disruption of zinc fingers 4, 5 or 6 had minimal effect on the DNA binding and TF activities of TFIIIA.

Present address: Owen Rowland, Department of Biology and Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada, K1S 5B6


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