Journal of Molecular Biology
CommunicationAssembly Architecture and DNA Binding of the Bacteriophage P22 Terminase Small Subunit
Section snippets
Preparation and purification of gp3 constructs
The plasmid construct employed for overexpression of full-length wild-type gp3 in Escherichia coli, the methods used for isolation and purification of the recombinant protein, and the demonstration of DNA packaging activity in Salmonella infections have been described.26 The full-length construct contained a 20-amino-acid N-terminal addition consisting of a histidine tag (His6) and thrombin cleavage site encoded by the pET-15b expression vector plasmid. The N-terminal addition was removed prior
Locus of the nonspecific DNA binding activity of gp3
DNA binding properties of the gp3 nonamer in the presence of a 50-bp DNA target that incorporated either the native 21-bp pac site or a functionally inactive permuted pac site were assayed previously by native gel electrophoresis.26 Similar gp3 binding affinities were observed for both DNA targets, indicating the formation of nonspecific gp3/DNA complexes. This is consistent with the expectation that genome translocation requires of terminase a nonspecific mode of DNA recognition in addition to
The fold of C-terminally truncated gp3
Raman spectra of nonameric assemblies of full-length and C-terminally truncated gp3 (lacking either 20 [gp3(Δ1–142)] or 10 [gp3(Δ1–152)] residues) exhibit very similar amide I (1658 cm− 1) and amide III (1253 cm− 1) markers, as shown in Fig. 2a. This suggests that the subunit fold is largely conserved after removal of residues 143–162. Quantitative analysis27 of the amide I band profile of the gp3(Δ1–142) truncate indicates 39 ± 2% α-helix, 21 ± 2% β-strand, and 40 ± 3% irregular structure, which
Three-dimensional structure of the gp3 assembly
Recently, we reported that the terminase small subunit of P22 assembles into a highly stable and symmetric ring.26 Two-dimensional averaging of the gp3 assemblies identified in electron micrographs of negatively stained particles revealed a central hole of approximately 20 Å in diameter with electron density emerging radially from the central annulus. Here, we have further analyzed the structure of the gp3 ring assembly by three-dimensional single-particle analysis of negatively stained gp3
Location of the gp3/DNA interface
Fig. 1, Fig. 2, Fig. 3 show that the excision of gp3 residues 143–162 eliminates nonspecific DNA binding, but does not impact either the native subunit fold of residues 1–142 or the overall nonameric ring structure that is visualized by negative-stain electron microscopy (EM). We attempted to further localize the nonspecific DNA-binding interface of gp3, within the context of the ring assembly, by computing a protein density difference map between rings consisting of full-length gp3 and the
Function of gp3 in the DNA packaging machine
The overall dimensions of the reconstruction of Fig. 3a are given in Table 1. These parameters suggest intriguing possibilities for DNA binding, pac site recognition, and genome translocation. For example, the central hole of the gp3 ring exhibits a diameter that is appropriate to the passage of dsDNA. It could thus provide a DNA-translocating channel that is dimensionally compatible with the translocating channel of the procapsid portal. The likely location of gp3 sites of nonspecific DNA
Acknowledgements
Support of this research by National Institutes of Health (NIH) grants GM50776 (G.J.T.) and AI074825 (S.R.C.) is gratefully acknowledged. Electron microscopic imaging and reconstruction were conducted at the National Resource for Automated Molecular Microscopy, which is supported by the NIH through a P41 program grant (RR17573) from the National Center for Research Resources. We are grateful to Drs. Bridget Carragher and Clint Potter for their interest and helpful discussions during EM
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Strategies for purification of the bacteriophage HK97 small and large terminase subunits that yield pure and homogeneous samples that are functional
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Large terminase conformational change induced by connector binding in bacteriophage T7
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2013, VirologyCitation Excerpt :The apparent flexibility of this connection is somewhat surprising, since it might have been expected that the different components of a DNA translocating molecular motor would have to occupy very specific spatial positions in the motor. However, there are other indications of TerS flexibility which include a mutant of P22 whose isolated TerS is present as decamers instead of nonamers that is functional in vivo (Nemecek et al., 2008), P22 TerS has 23 C-terminal amino acids that are not ordered in crystals (Roy et al., 2012), and analyses of alternate crystal structures of each of the SF6, T4 and Sf6 TerS proteins indicate that they have considerable structural flexibility (Buttner et al., 2012; Sun et al., 2012; Zhao et al., 2012). We also found that the N-terminal domain of TerS is responsible for the distribution of packaging series initiation DNA cleavages, since a P22 that carries only the DNA-binding domain of Sf6 TerS has a packaging initiation end distribution similar to that of phage Sf6 and not to that of phage P22.
Headful DNA packaging: Bacteriophage SPP1 as a model system
2013, Virus Research