Self-association properties of the bacteriophage lambda terminase holoenzyme: implications for the DNA packaging motor.
Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of viral DNA into a protective capsid. Bacteriophage lambda terminase holoenzyme is a hetero-oligomer composed of the A and Nu1 lambda gene products; however, the self-association properties of the holoenzyme have not been investigated systematically. Here, we report the results of sedimentation velocity, sedimentation equilibrium, and gel-filtration experiments studying the self-association properties of the holoenzyme. We find that purified, recombinant lambda terminase forms a homogeneous, heterotrimeric structure, consisting of one gpA molecule associated with two gpNu1 molecules (114.2 kDa). We further show that lambda terminase adopts a heterogeneous mixture of higher-order structures, with an average molecular mass of 528(+/-34) kDa. Both the heterotrimer and the higher-order species possess site-specific cos cleavage activity, as well as DNA packaging activity; however, the heterotrimer is dependent upon Escherichia coli integration host factor (IHF) for these activities. Furthermore, the ATPase activity of the higher-order species is approximately 1000-fold greater than that of the heterotrimer. These data suggest that IHF bending of the duplex at the cos site in viral DNA promotes the assembly of the heterotrimer into a biologically active, higher-order packaging motor. We propose that a single, higher-order hetero-oligomer of gpA and gpNu1 functions throughout lambda development.
Maluf
Nasib K
NK
Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, 4200 East Ninth Ave, C238, Denver, CO 80262, USA.
Yang
Qin
Q
Catalano
Carlos Enrique
CE
D000251:Adenosine Triphosphatases / Q000378:metabolism; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D042003:DNA Packaging; D004279:DNA, Viral / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008957:Models, Genetic; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000378:metabolism; D008970:Molecular Weight; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
D004279:DNA, Viral; D020035:Holoenzymes; D020409:Molecular Motor Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C030894:terminase; D000251:Adenosine Triphosphatases