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The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3 and E, and the two Escherichia coli genes groEL and groES . It has been shown previously that extracts prepared from cells infected with a lambda C-E- mutant accumulate a gpB polymer, which behaves as a biologically active intermediate in prohead assembly. This gpB activity has been called a preconnector , as it is probably a precursor to the head-tail connector. We now report the partial purification of biologically active preconnectors and the characterization of its structure. In the electron microscope, preconnectors appear as donut -like structures composed of several subunits displaying radial symmetry. Optical filtration of periodic arrays of preconnectors showed that the structure has 12-fold rotational symmetry. Side views of the preconnector reveal that it resembles an asymmetrical dumbell . This information has been used to construct a three-dimensional model of the preconnector . The implications of this structure for prohead shape and function, and for DNA packaging are discussed.

We have developed a new DNA-DNA crosslinking strategy based on a cleavable bispsoralen reagent and used this strategy to study the structures of bacteriophage lambda and the animal virus SV40. Our results show that in both lambda and SV40, all restriction fragments examined can be crosslinked to all other restriction fragments. In bacteriophage lambda, the crosslinking data are consistent with a random packaging model, while in SV40 there is some deviation from the random model. These results imply that the structures of DNA inside these viruses are either highly disordered or very complex.

Experiments are reported which bear on two spool models proposed for packaging the DNA of phage lambda. Both spool models fill an assumed spherical cavity with DNA wrapped in cylindrical or quasi-cylindrical layers composed of adjacent circular turns. In the curved-spool model, a single continuous segment of DNA, about 20% of the DNA length and probably located near the left end of the DNA, is in contact with the coat protein of the phage capsid. In the straight spool model, there are several DNA segments in contact with the capsid; they are concentrated in one half (probably the left half) of lambda DNA. We have identified the loci on the DNA which are in contact with the capsid by chemical crosslinking, induced by ultraviolet-irradiation of phage containing 5-bromodeoxyuridine in place of thymine. In an electron microscope experiment, phage are first lysed with EDTA, and then spread in a cytochrome c film by the formamide method. The disrupted capsid, which has the appearance of a phage ghost, serves as a marker showing where the DNA is crosslinked to the coat. The left end of the DNA is not distinguished from the right end, and so the map of DNA-capsid contacts is folded over on itself. Contacts are found nearly randomly over the entire map. In a second experiment, DNA from lysed, crosslinked phage is cut either with EcoRI or HindIII restriction endonucleases and the cut restriction fragments are labeled at their ends with 32P. Density centrifugation in a CsCl gradient separates free DNA from restriction fragments crosslinked to protein. After digestion with proteinase k, the DNA fragments previously crosslinked to protein are identified by size after agarose gel electrophoresis. DNA fragments from all parts of the genome are found. These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spool model is unlikely. Alternatively, the manner of packaging the DNA may vary from one phage to the next. These results agree with other recent experiments on lambda DNA packaging by Hall & Schellman (1982a,b), and by Haas et al. (1982). A different experiment is also reported. The psoralen derivative aminomethyltrioxalen (AMT) is allowed to intercalate into lambda phage and then the DNA strands are crosslinked by ultraviolet-irradiation after the rapid phase of AMT intercalation is complete. The DNA is subsequently denatured by glyoxal modification and spread for electron microscopy in a cytochrome c film by the formamide method.(ABSTRACT TRUNCATED AT 400 WORDS)

The three-dimensional structures of cro repressor protein and of the amino-terminal domain of lambda repressor protein, both from bacteriophage lambda, are compared. The second and third alpha-helices, alpha 2 and alpha 3, are shown to have essentially identical conformations in the two proteins, confirming the significance of the amino acid sequence homology previously noted between these and other DNA binding proteins in the region corresponding to these helices. The correspondence between the two-helical units in cro and lambda repressor protein is better than the striking agreement noted previously between two-helical units in cro and catabolite gene-activator protein. Parts of the first alpha-helices of repressor and cro show a structural correspondence that suggests a revised sequence homology between the two proteins in their extreme amino-terminal regions. In particular, there is a short loop between the alpha 1 and alpha 2 helices of lambda repressor that is missing from cro. This structural difference may account for the observed differences found with different cros and repressors in the pattern of phosphates whose ethylation prevents the binding of these proteins to their specific recognition sites. Although the two proteins have strikingly similar alpha 2-alpha 3 helical units that are presumed to bind to DNA in an essentially similar manner, stereochemical restrictions prevent the alpha 2-alpha 3 units of the respective proteins aligning on the DNA in exactly the same way.

The resonances of all the base protons and most of the sugar protons in both strands of the 17 base-pair OR3 operator of the phage lambda, and of the vC3 single base-pair mutant, have been assigned using two-dimensional nuclear magnetic resonance methods. The chemical shift and nuclear Overhauser effect data for these two DNA sequences reveal no structural perturbation at sites distal to the mutation, neither are there significant changes in structure immediately surrounding the altered base-pair in the mutant sequence. These results are consistent with the model proposed by Ohlendorf et al. (1982), based on crystallographic data on the cro protein, for the OR3-cro protein interaction. The data from these solution studies are examined and discussed in the light of this model. This work demonstrates that nuclear magnetic resonance chemical shifts and nuclear Overhauser effect intensities provide a method for comparing the solution structures of DNA molecules. From the resolution available in the spectra of the 17 base-pair operators studied, it is clear that DNA duplexes of up to 30 or more base-pairs can be studied using phase-sensitive methods.

The head shell of bacteriophage lambda expands by about 20% in diameter when it packages the DNA molecule in vivo. The expansion reaction is essentially a conformational change of the major head protein molecules to a state of lower free energy and can also be triggered in vitro by treatment with 4 M-urea. In order to investigate the conformational change, we have measured the circular dichroism, fluorescence and difference absorption spectra of the lambda head shell before and after the expansion by the treatment with urea. The far-ultraviolet circular dichroism spectra and the fluorescence spectra show that the expansion is not accompanied by a great change in the secondary structure (29% alpha-helix, 23% beta-structure) and the environment (non-polar) of the tryptophan residues of the major head protein molecule. On the other hand, by measurements of the circular dichroism and difference absorption spectra in the near-ultraviolet region as well as by chemical modification experiments with tetranitromethane, we have found that one or two tyrosine residues of the major head protein are transferred from a polar, solvent-exposed to a non-polar, solvent-unexposed environment during the expansion. Judging from these results, the conformational change seems to be mainly intermolecular or interdomainal rather than intradomainal.