Specificity of mammalian Y-box binding protein p50 in interaction with ss and ds DNA analyzed with generic oligonucleotide microchip.
p50 protein is a member of the Y-box binding transcription factor family and is a counterpart of YB-1 protein. The generic microchip was used to analyze the sequence specificity of p50 binding to single (ss) and double-stranded (ds) oligodeoxyribonucleotides. The generic microchip contained 4,096 single-stranded octadeoxyribonucleotides in which all possible core 6-mers (4(6)=4,096) were flanked at their 3' and 5'-ends with degenerated nucleotides. The oligonucleotides were chemically immobilized within polyacrylamide gel pads fixed on a glass slide. The binding of p50 to the generic microchip was shown to be the most specific to ss GGGG motif and then to ss CACC and CATC motifs. GC-rich ds oligonucleotides of the generic microchip, and particularly those containing GGTG/CACC, GATG/CATC, and GTGG/CCAC heterogeneous motifs, were most efficiently destabilized due to interaction with p50. Gel-shift electrophoresis has shown that the protein exhibits much higher binding specificity to 24-mer oligoA-TGGGGG-oligoA containing G-rich 6-mer, in comparison with 24-mer oligoA-AAATAT-oligoA carrying A,T-rich 6-mer in full correspondence with the data obtained with the microchip. Studies of DNA-binding proteins using gel-immobilized ss and ds DNA fragments provide a unique possibility to detect low-affinity complexes of these proteins with short sequence motifs and assess the role of these motifs in sequence-specific interactions with long recognition sites.
Zasedateleva
O A
OA
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, 119991 Moscow, Russian Federation.
Krylov
A S
AS
Prokopenko
D V
DV
Skabkin
M A
MA
Ovchinnikov
L P
LP
Kolchinsky
A
A
Mirzabekov
A D
AD
D000818:Animals; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D007091:Image Processing, Computer-Assisted; D008322:Mammals; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C118824:p50 Y-box-binding protein, mammalian; D004247:DNA