Visualization of two binding sites for the Escherichia coli UmuD'(2)C complex (DNA pol V) on RecA-ssDNA filaments.

The heterotrimeric UmuD'(2)C complex of Escherichia coli has recently been shown to possess intrinsic DNA polymerase activity (DNA pol V) that facilitates error-prone translesion DNA synthesis (SOS mutagenesis). When overexpressed in vivo, UmuD'(2)C also inhibits homologous recombination. In both activities, UmuD'(2)C interacts with RecA nucleoprotein filaments. To examine the biochemical and structural basis of these reactions, we have analyzed the ability of the UmuD'(2)C complex to bind to RecA-ssDNA filaments in vitro. As estimated by a gel retardation assay, binding saturates at a stoichiometry of approximately one complex per two RecA monomers. Visualized by cryo-electron microscopy under these conditions, UmuD'(2)C is seen to bind uniformly along the filaments, such that the complexes are completely submerged in the deep helical groove. This mode of binding would impede access to DNA in a RecA filament, thus explaining the ability of UmuD'(2)C to inhibit homologous recombination. At sub-saturating binding, the distribution of UmuD'(2)C complexes along RecA-ssDNA filaments was characterized by immuno-gold labelling with anti-UmuC antibodies. These data revealed preferential binding at filament ends (most likely, at one end). End-specific binding is consistent with genetic models whereby such binding positions the UmuD'(2)C complex (pol V) appropriately for its role in SOS mutagenesis.

Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD.

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved at 1.1 A resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0. 132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 A resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.

Engineering trimeric fibrous proteins based on bacteriophage T4 adhesins.

The adsorption specificity of bacteriophage T4 is determined by genes 12 and 37, encoding the short tail-fibers (STF) and the distal part of the long tail-fibers (LTF), respectively. Both are trimeric proteins with rod domains made up of similar tandem quasi-repeats, approximately 40 amino acids long. Their assembly requires the viral chaperones gp57A and gp38. Here we report that fusing fragments of gp12 and gp37 to another trimeric T4 fibrous protein, fibritin, facilitates correct assembly, thereby by-passing the chaperone requirement. Fibritin is an alpha-helical coiled coil protein whose C-terminal part (fibritin E, comprising the last 120 residues) has recently been solved to atomic resolution. Gp12 fragments of 109 and 70 amino acids, corresponding to three and two quasi-repeats respectively, were fused to the C-terminus of fibritin E. A similar chimera was designed for the last 63 residues of gp37, which contain four copies of the pentapeptide Gly-X-His-X-His and assume a narrow rigid structure in the LTF distal tip. Expressed from plasmids, all three chimeras form soluble trimers that are resistant to dissociation by SDS and digestion by trypsin, indicative of correct folding and oligomerization.

Encapsidated conformation of bacteriophage T7 DNA.

The structural organization of encapsidated T7 DNA was investigated by cryo-electron microscopy and image processing. A tail-deletion mutant was found to present two preferred views of phage heads: views along the axis through the capsid vertex where the connector protein resides and via which DNA is packaged; and side views perpendicular to this axis. The resulting images reveal striking patterns of concentric rings in axial views, and punctate arrays in side views. As corroborated by computer modeling, these data establish that the T7 chromosome is spooled around this axis in approximately six coaxial shells in a quasi-crystalline packing, possibly guided by the core complex on the inner surface of the connector.

Stoichiometry and domainal organization of the long tail-fiber of bacteriophage T4: a hinged viral adhesin.

The long-tail fibers (LTFs) form part of bacteriophage T4's apparatus for host cell recognition and infection, being responsible for its initial attachment to susceptible bacteria. The LTF has two parts, each approximately 70 to 75 nm long; gp34 (140 kDa) forms the proximal half-fiber, while the distal half-fiber is composed of gp37 (109 kDa), gp36(23 kDa) and gp35 (30 kDa). LTFs have long been thought to be dimers of gp34, gp37 and gp36, with one copy of gp35. We have used mass mapping by scanning transmission electron microscopy (STEM), quantitative SDS-PAGE, and computational sequence analysis to study the structures of purified LTFs and half-fibers of both kinds. These data establish that the LTF is, in fact, trimeric, with a stoichiometry of gp34: gp37: gp36: gp35 = 3:3:3:1. Averaged images of stained and unstained molecules resolve the LTF into a linear stack of 17 domains. At the proximal end is a globular domain of approximately 145 kDa that becomes incorporated into the baseplate. It is followed by a rod-like shaft (33 x 4 mm; 151 kDa) which correlates with a cluster of seven quasi repeats, each 34 to 39 residues long. The proximal half-fiber terminates in three globular domains. The distal half-fiber consists of ten globular domains of variable size and spacing, preceding a needle-like end domain (15 x 2.5 nm; 31 kDa). The LTF is rigid apart from hinges between the two most proximal domains, and between the proximal and distal half-fibers. The latter hinge occurs at a site of local non-equivalence (the "kneecap") at which density, correlated with the presence of gp35, bulges asymmetrically out on one side. Several observations indicate that gp34 participates in the sharing of conserved structural modules among coliphage tail-fiber genes to which gp37 was previously noted to subscribe. Two adjacent globular domains in the proximal half-fiber match a pair of domains in the distal half-fiber, and the rod domain in the proximal half-fiber resembles a similar domain in the T4 short tail-fiber (gp12). Finally, possible structures are considered; combining our data with earlier observations, the most likely conformation for most of the LTF is a three-stranded beta-helix.

Assembly of T7 capsids from independently expressed and purified head protein and scaffolding protein.

Prohead-like capsid shells containing the scaffolding and head proteins of bacteriophage T7 were isolated after both proteins were expressed from the cloned genes in the same cell. When the head-tail connector protein was also expressed, the isolated capsids contained neither connector nor scaffolding protein and resembled mature phage capsids rather than proheads. However, only a small fraction of the head protein was converted to stable capsid structures in either case. Purified scaffolding protein (expressed individually from the cloned gene) appeared to be a monomer in solution; purified head protein appeared to be a tetramer. The purified proteins reacted in the presence of polyethylene glycol or dextran to produce prohead-like capsid shells and also polycapsids consisting primarily of head protein, similar to the polycapsids observed after infection by T7 mutants lacking connector or core proteins. Neither capsids nor polycapsids were produced in the absence of scaffolding protein. Polycapsids were usually the predominant product even when scaffolding protein was in excess, and a small fraction of scaffolding protein catalyzed the conversion of an excess of head protein to polycapsids. Our results suggest that the first step in the natural pathway to prohead formation is the assembly of incomplete prohead shells, which are normally closed by insertion of a connector-core complex. In the absence of a functional connector-core complex, incomplete capsid shells apparently react further to form polycapsids or completely closed capsid shells.

Purification and characterization of T7 head-tail connectors expressed from the cloned gene.

Cloned gene 8, which specifies the protein of the head-tail connector of bacteriophage T7, was expressed in Escherichia coli. Extracts prepared in a low-salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates. Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography perhaps because of dissociation of monomer-connector complexes or disassembly of unstable connectors. Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography but not before. Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography. The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis. Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay. No ATPase activity was detected in either monomer or connector preparations in the absence or presence of DNA.

Improved methods for determination of rotational symmetries in macromolecules.

Rotational symmetries of macromolecules are most clearly perceived in the en face projection and may be assessed by inspection of rotational power spectra calculated from electron micrographs of individual particles. However, if the symmetry is not contrasted strongly, this procedure may be inconclusive since the relevant peak may not be convincingly higher than other spectral components. To some extent, this is a sampling problem since the number of repeating elements involved is usually small. We have devised more sensitive statistical tests for rotational symmetry that pool the information contents of entire populations of particles. Both tests involve combining the rotational spectra of many particles and comparing them with the spectra of surrounding background areas. One method is based on the well known t-test which estimates whether two populations differ at a given significance level. In the second test, the ratio between the intensity of each component of the rotational spectrum and the average corresponding intensity for background areas is calculated, and thence, the cumulative product of these ratios over all particles in the data set. If a symmetry is present, this product gradually diverges; otherwise, it converges to zero. As a practical trial, the tests were applied to micrographs of negatively stained hexons of herpes simplex virus and confirmed their 6-fold symmetry. Applied to negatively stained "connector" proteins of bacteriophage T7 purified from a plasmid expression system, both algorithms detected polymorphism with distinct subpopulations of both 13-fold and 12-fold connectors.