Pentaprobe: a comprehensive sequence for the one-step detection of DNA-binding activities.
The rapid increase in the number of novel proteins identified in genome projects necessitates simple and rapid methods for assigning function. We describe a strategy for determining whether novel proteins possess typical sequence-specific DNA-binding activity. Many proteins bind recognition sequences of 5 bp or less. Given that there are 4(5) possible 5 bp sites, one might expect the length of sequence required to cover all possibilities would be 4(5) x 5 or 5120 nt. But by allowing overlaps, utilising both strands and using a computer algorithm to generate the minimum sequence, we find the length required is only 516 base pairs. We generated this sequence as six overlapping double-stranded oligonucleotides, termed pentaprobe, and used it in gel retardation experiments to assess DNA binding by both known and putative DNA-binding proteins from several protein families. We have confirmed binding by the zinc finger proteins BKLF, Eos and Pegasus, the Ets domain protein PU.1 and the treble clef N- and C-terminal fingers of GATA-1. We also showed that the N-terminal zinc finger domain of FOG-1 does not behave as a typical DNA-binding domain. Our results suggest that pentaprobe, and related sequences such as hexaprobe, represent useful tools for probing protein function.
Kwan
Ann H Y
AH
School of Molecular and Microbial Biosciences, G08, University of Sydney, NSW 2006, Australia.
Czolij
Robert
R
Mackay
Joel P
JP
Crossley
Merlin
M
D000465:Algorithms; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D004247:DNA