Bettina Wolpensinger

Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC

BACKGROUND DNA helicases play a fundamental role in all aspects of nucleic acid metabolism and defects in these enzymes have been implicated in a number of inherited human disorders. DnaB is the major replicative DNA helicase in Escherichia coli and has been used as a model system for studying the structure and function of hexameric helicases. The native protein is a hexamer of identical subunits, which in solution forms a complex with six molecules of the loading protein DnaC. DnaB is delivered from this complex onto the DNA template, with the subsequent release of DnaC. We report here the structures of the DnaB helicase hexamer and its complex with DnaC under a defined set of experimental conditions, as determined by three-dimensional cryoelectron microscopy. It was hoped that the structures would provide insight into the mechanisms of helicase activity. RESULTS The DnaB structure reveals that six DnaB monomers assemble as three asymmetric dimers to form a polar, ring-like hexamer. The hexamer has two faces, one displaying threefold and the other sixfold symmetry. The six DnaC protomers bind tightly to the sixfold face of the DnaB hexamer. This is the first report of a three-dimensional structure of a helicase obtained using cryoelectron microscopy, and the first report of the structure of a helicase in complex with a loading protein. CONCLUSIONS The structures of the DnaB helicase and its complex with DnaC reveal some interesting structural features relevant to helicase function and to the assembly of the two-protein complex. The results presented here provide a basis for a more complete understanding of the structure and function of these important proteins.

Analysis of the photosystem II reaction centers using scanning transmision electron microscopy

Photosynthetic RCs are classified according to the nature of their terminal electron acceptors. Q-type RCs (type II) reduce a mobile quinone. Examples are found in purple bacteria and in the photosystem II (PSII) of cyanobacteria and higher plants [1]. The RC of PSII consists of the PsbA, PsbD, PsbE, PsbF and PsbI subunits. The PsbA/PsbD heterodimer binds a variety of cofactors. These include the chlorophylls of the primary donor P680, the primary acceptor pheophytin, QA, the non-heme iron, QB, and the radicals TyrZ+ and TyrD+. Four additional chlorophylls, the “non-photochemical” pheophytin, and one or two s-carotene are also bound. A 9 kDa and a 5 kDa membrane protein, the psbE and psbF gene products respectively, form the cyt b559 which is required for the PSII assembly. Cyt b559 is thought to be involved in cyclic electron flow around PSII since it has been shown to be both, photooxydized by P680+ and photoreduced by plastoquinol [2]. The function of the small 4 kDa membrane subunit (psbI gene product) also present is not know. In the present communication we report structural studies of a RC complex containing the PsbA, PsbD, PsbE, PsbF and PsbI subunits from spinach. Mass data and structural information was obtained by gel filtration and scanning transmission electron microscopy (STEM). The structural information gained from this PSII subcomplex allowed the other PSII complex components to be localized in the structure.