Specific interaction and two-dimensional crystallization of histidine tagged yeast RNA polymerase I on nickel-chelating lipids.
Nickel-chelating lipid monolayers were used to generate two-dimensional crystals from yeast RNA polymerase I that was histidine-tagged on one of its subunits. The interaction of the enzyme with the spread lipid layers was found to be imidazole dependent, and the formation of two-dimensional crystals required small amounts of imidazole, probably to select the specific interaction of the engineered tag with the nickel. Two distinct preparations of RNA polymerase I tagged on different subunits yielded two different crystal forms, indicating that the position of the tag determines the crystallization process. The orientation of the enzyme in both crystal forms is correlated with the location of the tagged subunits in a three-dimensional model which shows that the tagged subunits are in contact with the lipid layer.
Bischler
N
N
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP 1, C.U. de Strasbourg, France.
Balavoine
F
F
Milkereit
P
P
Tschochner
H
H
Mioskowski
C
C
Schultz
P
P
D002614:Chelating Agents; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D019281:Dimerization; D006639:Histidine; D008055:Lipids; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008958:Models, Molecular; D009532:Nickel; D010455:Peptides; D011487:Protein Conformation; D012318:RNA Polymerase I / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016324:Sequence Tagged Sites
C109930:2-(bis-carboxymethylamino)-6-(2-(1,3-di-O-oleylglyceroxy)acetylamino)hexanoic acid nickel(II); D002614:Chelating Agents; D008055:Lipids; D046911:Macromolecular Substances; D010455:Peptides; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D009532:Nickel; D012318:RNA Polymerase I