Journal of Molecular Biology
Volume 383, Issue 3, 14 November 2008, Pages 494-501
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Assembly Architecture and DNA Binding of the Bacteriophage P22 Terminase Small Subunit

https://doi.org/10.1016/j.jmb.2008.08.050Get rights and content

Abstract

Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally encoded molecular motor called terminase, which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2. Electron microscopy shows that wild-type gp3 self-assembles into a stable and monodisperse nonameric ring. A three-dimensional reconstruction at 18 Å resolution provides the first glimpse of P22 terminase architecture and implies two distinct modes of interaction with DNA—involving a central channel of 20 Å diameter and radial spikes separated by 34 Å. Electromobility shift assays indicate that the gp3 ring binds double-stranded DNA nonspecifically in vitro via electrostatic interactions between the positively charged C-terminus of gp3 (residues 143–152) and phosphates of the DNA backbone. Raman spectra show that nonameric rings formed by subunits truncated at residue 142 retain the subunit fold despite the loss of DNA-binding activity. Difference density maps between gp3 rings containing full-length and C-terminally truncated subunits are consistent with localization of residues 143–152 along the central channel of the nonameric ring. The results suggest a plausible molecular mechanism for gp3 function in DNA recognition and translocation.

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

References (40)

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