ATPase center of bacteriophage lambda terminase involved in post-cleavage stages of DNA packaging: identification of ATP-interactive amino acids.
Terminase is the enzyme that mediates lambda DNA packaging into the viral prohead. The large subunit of terminase, gpA (641 amino acid residues), has a high-affinity ATPase activity (K(m)=5 microM). To directly identify gpA's ATP-interacting amino acids, holoterminase bearing a His(6)-tag at the C terminus of gpA was UV-crosslinked with 8-N(3)-[alpha-(32)P]ATP. Tryptic peptides from the photolabeled terminase were purified by affinity chromatography and reverse-phase HPLC. Two labeled peptides of gpA were identified. Amino acid sequencing failed to show the tyrosine residue of the first peptide, E(43)SAY(46)QEGR(50), or the lysine of the second peptide, V(80)GYSK(84)MLL(87), indicating that Y(46) and K(84) were the 8-N(3)-ATP-modified amino acids. To investigate their roles in lambda DNA packaging, Y(46) was changed to E, A, and F, and K(84) was changed to E and A. Purified His(6)-tagged terminases with changes at residues 46 and 84 lacked the gpA high-affinity ATPase activity, though the cos cleavage and cohesive end separation activities were near to those of the wild-type enzyme. In virion assembly reactions using virion DNA as a packaging substrate, the mutant terminases showed severe defects. In summary, the results indicate that Y(46) and K(84) are part of the high-affinity ATPase center of gpA, and show that this ATPase activity is involved in the post-cos cleavage stages of lambda DNA packaging.
Hang
J Q
JQ
College of Medicine, The Molecular Biology Program and Department of Microbiology, Iowa City, IA, 52242, USA.
Tack
B F
BF
Feiss
M
M
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001287:Attachment Sites, Microbiological / Q000235:genetics; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019920:Photoaffinity Labels; D023302:Protein Footprinting; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays; D014771:Virion / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D019065:Virus Assembly
D004279:DNA, Viral; D020035:Holoenzymes; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C030894:terminase; D014357:Trypsin; D000251:Adenosine Triphosphatases