Isolation and characterization of a protein with high affinity for DNA: the glutamine synthetase of Thermus thermophilus 111.

摘要

In a search of proteins from the thermophilic bacterium Thermus thermophilus 111 with a high affinity for DNA, the selected protein from this screening appears to be the glutamine synthetase (GS). The purified product gives one band in SDS-polyacrylamide gel electrophoresis (53,700 Da). The N-terminal 32 residues have been identified and present an homology of 80% with the glutamine synthetase of Bacillus subtilis and 76% with that of Thermotoga maritima. The protein displays the characteristic dodecameric structure of the eubacteria glutamine synthetase. From a detailed study of the interaction of this protein with DNA by dark-field electron microscopy and agarose gel electrophoresis, it is concluded that double-stranded DNA wraps the protein by a full turn of 150 bp length. An even number of GS molecules bound to a closed relaxed plasmid DNA does not alter its null topology. By using an inverted dimer DNA fragment, which contains twice a curved kinetoplast DNA insert in its central part, it is shown that DNA curvature rules the order in which GS binds to the DNA. DNA ends are also sites of high affinity for the GS. Supercoiling does not favor the binding of GS to the DNA with the exception of the apices that are by essence bent regions. By saturating a DNA molecule with GS one obtains a novel characteristic scalloped configuration in which the DNA undulates from one GS to the next. The DNA is condensed at least three times in these structures. By increasing the ratio of GS to DNA in solution the resulting material migrates as discrete bands relative to the free DNA in an agarose gel. By gel retardation and EM statistical distribution analysis of GS within the complexes, an average affinity constant of 10(7) M-1 was obtained. The potential implications of this novel interaction of the glutamine synthetase with DNA for the regulation of its own gene are briefly discussed. Copyright 1999 Academic Press.