DNA-binding properties of gene-5 protein encoded by bacteriophage M 13. 1. The kinetics of the dissociation of gene-5-protein.polynucleotide complexes upon addition of salt.
The irreversible dissociation kinetics of complexes of M13-encoded gene-5 protein with the polynucleotides poly(dA) and M13 DNA was studied by means of stopped-flow experiments. A linear decay was found for all gene-5-protein.poly(dA) complexes and for the gene-5-protein.M13 DNA complexes for which the DNA lattice was completely saturated at the beginning of the dissociation experiments. Only at the end of the dissociation curve was a deviation from linearity observed. A single-exponential decay was found for the dissociation of gene-5-protein.M13 DNA complexes when the DNA was not completely saturated initially. These results could be interpreted by assuming that dissociation of bound protein is only possible from isolated binding sites, while during the dissociation, rearrangement of bound protein clusters takes place continuously, including the formation of newly isolated bound protein. This redistribution results from a translocation of the protein along the lattice, which, for the poly(dA) complex, is fast with respect to the dissociation step, but which is slow for the M13 DNA complex. During this process the equilibrium cluster distribution predicted by the theory of McGhee and Von Hippel is not maintained. The binding of gene-5 protein to poly(dA) or poly(dT) does not result in a broadening of the nucleotide resonances in the NMR spectra of these polynucleotides, as had been observed for E. coli DNA-binding protein and interpreted as an indication for a high rate of translocation of the protein on the polynucleotide. The absence of line broadening for gene-5-protein.polynucleotide complexes is caused by the high binding cooperativity. As a consequence the majority of the protein molecules are bound in a cluster which makes the concentration of isolated bound protein very low. This results in a decrease of the signal/noise ratio at higher degrees of binding, but does not lead to line broadening while fast translocation still occurs.
Bulsink
H
H
Laboratory of Biophysical Chemistry, University of Nijmegen, The Netherlands.
Harmsen
B J
BJ
Hilbers
C W
CW
D001667:Binding, Competitive; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D011061:Poly A / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D012492:Salts; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011089:Polydeoxyribonucleotides; D012492:Salts; D014764:Viral Proteins; D011061:Poly A; C015465:poly(dA)