Plasmid vectors for the analysis of protein-induced DNA bending.
Bending is not only required to accommodate DNA within the cell but also is a mechanism used by proteins to initiate DNA replication, transcription, and recombination. Determining the angles by which regulatory DNA segments deviate from linearity upon binding of proteins is a necessary step toward a better understanding of a large number of essential biological functions. The pBend plasmids contain duplicate sets of restriction sites and, when combined with "gel shift" experiments, allow the straightforward determination of the bending angle in a DNA molecule. The steps for successfully carrying out a binding/bending experiment are described. They include the cloning of the protein-binding site into the chosen pBend vector, the isolation of a series of DNA fragments with identical in length but variable placing of the protein-binding site, and the gel electrophoretic analysis of the free and protein-bound fragments.
Department of Molecular Biology, The University of Texas Health Science, Center at Tyler, P.O. Box 2003, Tyler, TX 75710, USA.
Christian
C
Zwieb
Sankar
S
Adhya
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002999:Clone Cells; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D011506:Proteins / Q000378:metabolism* / Q000494:pharmacology
D002373:Cyclic AMP Receptor Protein; D011506:Proteins; D004247:DNA; D004262:DNA Restriction Enzymes