Assembly of a nucleoprotein complex required for DNA packaging by bacteriophage lambda.
A critical step in the assembly of bacteriophage lambda is the excision of a single genome from a concatemeric DNA precursor and insertion of genomic DNA into an empty viral capsid. DNA packaging is mediated by the lambda proteins gpNu1 and gpA, which form an enzyme complex known as terminase. Initiation of the packaging process requires assembly of the terminase subunits onto cos, the lambda DNA packaging sequence, and nicking of the duplex, thus forming the 12-base-pair "sticky" ends of the mature genome. We have utilized gel-retardation techniques to examine the interaction of gpNu1, gpA, and terminase holoenzyme with DNA. Our data demonstrate that gpNu1 interacts specifically with cos-containing DNA, forming three gel-retarded complexes. Similarly, the larger gpA subunit binds to DNA, forming two complexes; however, this subunit forms similar complexes with DNA substrates of random sequence. All of the nucleoprotein complexes examined are disrupted by elevated concentrations of NaCl and we suggest that altered DNA binding is responsible for the extreme salt sensitivity of the endonuclease activity of the enzyme [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065]. DNA binding by each subunit is strongly affected by the presence of the other, with 10- and 3-fold increases in the affinity of gpNu1 and gpA, respectively, for DNA. Moreover, our data suggest that the terminase subunits interact in solution prior to DNA binding. Finally, we provide evidence that complex I, the first stable intermediate in the packaging pathway, is composed of the mature left genome end bound to the terminase subunits and demonstrate that dissociation of the complex is quite slow (t1/2 > 8 h). The significance of these data with respect to terminase-mediated genome packaging is discussed.
Yang
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Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262, USA.
Hanagan
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Catalano
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D010582:Bacteriophage lambda / Q000378:metabolism / Q000502:physiology*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D009698:Nucleoproteins / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D014764:Viral Proteins; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C030894:terminase; D008274:Magnesium