Immobilization of biotinylated DNA on 2-D streptavidin crystals.
The structural study of transient nucleoprotein complexes by electron microscopy is hampered by the coexistence of multiple interaction states leading to an heterogeneous image population. To tackle this problem, we have investigated the controlled immobilization of double stranded DNA molecules and of nucleoprotein complexes onto a support suitable for cryo-electron microscopy observation. The DNA was end-labeled with a biotin moiety in order to decorate, or to be incorporated into, two-dimensional streptavidin crystals formed in contact of a biotinylated lipid layer. The binding specificity and efficiency were examined by radioactively labeled oligonucleotides and by direct visualization of unstained and hydrated nucleic acid molecules in cryo-electron microscopy. By using RNA polymerase we further show that, once immobilized, femtomolar amounts of DNA template are suitable to interact with the enzyme. The image analysis of the RNA polymerase-DNA complexes showed that a three-dimensional model can be retrieved from such samples.
Crucifix
Corinne
C
Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP 1, rue Laurent Fries, BP10142 67404 Illkirch, France.
Uhring
Muriel
M
Schultz
Patrick
P
D019921:Biotinylation; D020285:Cryoelectron Microscopy / Q000379:methods*; D003460:Crystallization; D004247:DNA / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D021621:Imaging, Three-Dimensional; D009698:Nucleoproteins / Q000737:chemistry; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry*
D009698:Nucleoproteins; D004247:DNA; D019809:Streptavidin; D012321:DNA-Directed RNA Polymerases