Effects of different zinc finger transcription factors on genomic targets

Effects of different zinc finger transcription factors on genomic targets We have developed a novel vector system for the efficient assembly of polydactyl zinc fingers. Next to proteins that possess short canonical TGEKP linkers between all constituting zinc fingers we constructed proteins with longer, 12 amino acid linkers between two three-finger (3F) units and between three two-finger (2F) units. Fusions of these zinc finger domains with the VP16 activation domain were tested for their ability to regulate a repressed genomic locus containing contiguous or noncontiguous zinc finger binding sites in yeast. In contrast to other studies, which were mostly confined to in vitro tests, we did not obtain evidence that superior artificial zinc finger transcription factors need to contain longer linkers between individual fingers. For the regulation of genomic loci, canonical linkers within a highly regular backbone in combination with a contiguous 18 base pair DNA target site were found to provide a sound base for polydactyl zinc finger design. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biochemical and Biophysical Research Communications Elsevier

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Publisher
Elsevier
Copyright
Copyright © 2005 Elsevier Inc.
ISSN
0006-291x
D.O.I.
10.1016/j.bbrc.2005.11.011
Publisher site
See Article on Publisher Site

Abstract

We have developed a novel vector system for the efficient assembly of polydactyl zinc fingers. Next to proteins that possess short canonical TGEKP linkers between all constituting zinc fingers we constructed proteins with longer, 12 amino acid linkers between two three-finger (3F) units and between three two-finger (2F) units. Fusions of these zinc finger domains with the VP16 activation domain were tested for their ability to regulate a repressed genomic locus containing contiguous or noncontiguous zinc finger binding sites in yeast. In contrast to other studies, which were mostly confined to in vitro tests, we did not obtain evidence that superior artificial zinc finger transcription factors need to contain longer linkers between individual fingers. For the regulation of genomic loci, canonical linkers within a highly regular backbone in combination with a contiguous 18 base pair DNA target site were found to provide a sound base for polydactyl zinc finger design.

Journal

Biochemical and Biophysical Research CommunicationsElsevier

Published: Jan 6, 2006

References

  • Solution structure of the first three zinc fingers of TFIIIA bound to the cognate DNA Sequence: determinants of affinity and sequence specificity
    Wuttke, D.S.; Foster, M.P.; Case, D.A.; Gottesfeld, J.M.; Wright, P.E.
  • Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds
    Mumberg, D.; Müller, R.; Funk, M.
  • Zero background reporter plasmids
    Melcher, K.; Sharma, B.; Ding, W.V.; Nolden, M.
  • Gal80–Gal80 interaction on adjacent Gal4p binding sites is required for complete GAL gene repression
    Melcher, K.; Xu, H.E.
  • Vectors for transcription factor cloning and target site identification by means of genetic selection in yeast
    Meijer, A.H.; Ouwerkerk, P.B.F.; Hoge, J.H.C.
  • Constraints for zinc finger linker design as inferred from X-ray crystal structure of tandem Zif268–DNA complexes
    Peisach, E.; Pabo, C.O.

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