Oligomeric states of bacteriophage T7 gene 4 primase/helicase.
Electron microscopic and crystallographic data have shown that the gene 4 primase/helicase encoded by bacteriophage T7 can form both hexamers and heptamers. After cross-linking with glutaraldehyde to stabilize the oligomeric protein, hexamers and heptamers can be distinguished either by negative stain electron microscopy or electrophoretic analysis using polyacrylamide gels. We find that hexamers predominate in the presence of either dTTP or beta,gamma-methylene dTTP whereas the ratio between hexamers and heptamers is nearly the converse in the presence of dTDP. When formed, heptamers are unable to efficiently bind either single-stranded DNA or double-stranded DNA. We postulate that a switch between heptamer to hexamer may provide a ring-opening mechanism for the single-stranded DNA binding pathway. Accordingly, we observe that in the presence of both nucleoside di- and triphosphates the gene 4 protein exists as a hexamer when bound to single-stranded DNA and as a mixture of heptamer and hexamer when not bound to single-stranded DNA. Furthermore, altering regions of the gene 4 protein postulated to be conformational switches for dTTP-dependent helicase activity leads to modulation of the heptamer to hexamer ratio.
Crampton
Donald J
DJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. dcrampton@hms.harvard.edu
Ohi
Melanie
M
Qimron
Udi
U
Walz
Thomas
T
Richardson
Charles C
CC
D001120:Arginine / Q000378:metabolism; D017123:Bacteriophage T7 / Q000201:enzymology*; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006868:Hydrolysis; D008954:Models, Biological; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
D004277:DNA, Single-Stranded; D009711:Nucleotides; D006639:Histidine; D001120:Arginine; D019915:DNA Primase