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.
Neuteboom
Leon W
LW
Institute of Biology Leiden, Department of Molecular and Developmental Genetics, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.
Lindhout
Beatrice I
BI
Saman
Ingrid L
IL
Hooykaas
Paul J J
PJ
van der Zaal
Bert J
BJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors