Imaging the asymmetrical DNA bend induced by repressor activator protein 1 with scanning tunneling microscopy.
The yeast Repressor Activator Protein 1 (RAP1) binds a 13-bp consensus found in many transcriptional regulatory regions, in silencer elements, and in telomeric repeat DNA of Saccharomyces cerevisiae. Gel retardation assays suggest that RAP1 bends DNA as it binds, with the vertex of the angle located 5' of the consensus. We show that removal of 230 aa in the N-terminus of RAP1 reduces the aberrant electrophoretic mobility of the protein-DNA complex, while removal of a C-terminal domain of RAP1 causes even greater distortion. To demonstrate that the aberrant electrophoretic mobility is really due to a bend in the double helix, the RAP1-DNA complex was analyzed by Scanning Tunnelling Microscopy (STM). The efficiency and accuracy of binding is checked in parallel by standard Transmission Electron Microscopy (TEM). Due to the use of high-angle shadowing of freeze-dried samples at low temperatures, the STM images allow us to confirm that RAP1 binding induces a DNA bend > 50 degrees, while the binding of the minimal DNA-binding domain shows significantly less distortion of the DNA helix.
Müller
T
T
Eidgenössische Technische Hochschule, Institut für Zellbiologie, Zürich, Switzerland.
Gilson
E
E
Schmidt
R
R
Giraldo
R
R
Sogo
J
J
Gross
H
H
Gasser
S M
SM
D004247:DNA / Q000648:ultrastructure*; D005612:Freeze Drying; D005656:Fungal Proteins / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods; D016252:Microscopy, Scanning Tunneling / Q000379:methods; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000648:ultrastructure*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D020668:rap GTP-Binding Proteins
D005656:Fungal Proteins; D011994:Recombinant Proteins; D004247:DNA; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins