Three-dimensional electron microscopy of the clamp loader small subunit from Pyrococcus furiosus.
An archaeal clamp loader, replication factor C (RFC), consists of two proteins, the small subunit (RFCS) and large subunit (RFCL), whose sequences are both highly homologous to those of the eukaryotic RFC components. We have investigated the oligomeric structure of RFCS from Pyrococcus furiosus by electron microscopy using single-particle analysis. RFCS forms mostly ring-shaped hexamers at pH 9.0, although it tends to form C-shaped tetramers or pentamers at a lower pH (pH 5.5). The three-dimensional (3D) structure of the RFCS hexamer was obtained by random conical tilt reconstruction at 24.0-A resolution. RFCS forms a hexameric ring with outer and inner diameters of 117 and 27 A, respectively, and with a height of about 55 A. The six subunits are arranged in a twisted manner with a sixfold symmetry around the channel. The 3D map revealed that the six subunits are arranged in a head-to-tail configuration. Although the RFC complex consists of RFCS and RFCL in vivo, RFCS alone, together with PCNA, substantially enhanced the DNA synthesizing activity of P. furiosus DNA polymerase I in vitro. The 3D reconstruction of RFCS with catalytic activity provides important insights into the organization mechanism and the functional state of the RFC complex.
Mayanagi
K
K
Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita-City, 565-0874, Japan. maya@beri.co.jp
Miyata
T
T
Oyama
T
T
Ishino
Y
Y
Morikawa
K
K
D019843:Archaeal Proteins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000494:pharmacology; D004256:DNA Polymerase I / Q000187:drug effects / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D021122:Protein Subunits; D011506:Proteins / Q000737:chemistry* / Q000494:pharmacology; D020027:Pyrococcus furiosus / Q000737:chemistry*; D051818:Replication Protein C
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D021122:Protein Subunits; D011506:Proteins; C424709:rfcL protein, Pyrococcus furiosus; C424708:rfcS protein, Pyrococcus furiosus; D004256:DNA Polymerase I; D051818:Replication Protein C