Sequence specificity of single-stranded DNA-binding proteins: a novel DNA microarray approach.
We have developed a novel DNA microarray-based approach for identification of the sequence-specificity of single-stranded nucleic-acid-binding proteins (SNABPs). For verification, we have shown that the major cold shock protein (CspB) from Bacillus subtilis binds with high affinity to pyrimidine-rich sequences, with a binding preference for the consensus sequence, 5'-GTCTTTG/T-3'. The sequence was modelled onto the known structure of CspB and a cytosine-binding pocket was identified, which explains the strong preference for a cytosine base at position 3. This microarray method offers a rapid high-throughput approach for determining the specificity and strength of ss DNA-protein interactions. Further screening of this newly emerging family of transcription factors will help provide an insight into their cellular function.
Morgan
Hugh P
HP
Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, UK.
Estibeiro
Peter
P
Wear
Martin A
MA
Max
Klaas E A
KE
Heinemann
Udo
U
Cubeddu
Liza
L
Gallagher
Maurice P
MP
Sadler
Peter J
PJ
Walkinshaw
Malcolm D
MD
D019843:Archaeal Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002151:Calorimetry; D002232:Carbocyanines; D016384:Consensus Sequence; D003596:Cytosine / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins / Q000378:metabolism; D008958:Models, Molecular; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D002232:Carbocyanines; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; C407094:cold shock protein, Bacillus; C085321:cyanine dye 5; D003596:Cytosine