Hierarchical mechanisms build the DNA-binding specificity of FUSE binding protein.
The far upstream element (FUSE) binding protein (FBP), a single-stranded nucleic acid binding protein, is recruited to the c-myc promoter after melting of FUSE by transcriptionally generated dynamic supercoils. Via interactions with TFIIH and FBP-interacting repressor (FIR), FBP modulates c-myc transcription. Here, we investigate the contributions of FBP's 4 K Homology (KH) domains to sequence selectivity. EMSA and missing contact point analysis revealed that FBP contacts 4 separate patches spanning a large segment of FUSE. A SELEX procedure using paired KH-domains defined the preferred subsequences for each KH domain. Unexpectedly, there was also a strong selection for the noncontacted residues between these subsequences, showing that the contact points must be optimally presented in a backbone that minimizes secondary structure. Strategic mutation of contact points defined in this study disabled FUSE activity in vivo. Because the biological specificity of FBP is tuned at several layers: (i) accessibility of the site; (ii) supercoil-driven melting; (iii) presentation of unhindered bases for recognition; and (iv) modular interaction of KH-domains with cognate bases, the FBP-FIR system and sequence-specific, single-strand DNA binding proteins in general are likely to prove versatile tools for adjusting gene expression.
Benjamin
Lawrence R
LR
Gene Regulation Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Building 10, Room 2N106, Bethesda, MD 20892-1500, USA.
Chung
Hye-Jung
HJ
Sanford
Suzanne
S
Kouzine
Fedor
F
Liu
Juhong
J
Levens
David
D
D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D016384:Consensus Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D016259:Genes, myc; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016601:RNA-Binding Proteins; D052156:SELEX Aptamer Technique
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C492258:FUBP1 protein, human; D016601:RNA-Binding Proteins; D004265:DNA Helicases