Interactions of the Streptomyces lividans initiator protein DnaA with its target.
The Streptomyces lividans DnaA protein (73 kDa) consists, like other bacterial DnaA proteins, of four domains; it binds to 19 DnaA boxes in the complex oriC region. The S. lividans DnaA protein differs from others in that it contains an additional stretch of 120 predominantly acidic amino acids within domain II. Interactions between the DnaA protein and the two DnaA boxes derived from the promoter region of the S. lividans dnaA gene were analysed in vitro using three independent methods: Dnase-I-footprinting experiments, mobility-shift assay and surface plasmon resonance (SPR). The Dnase-I-footprinting analysis showed that the wild-type DnaA protein binds to both DnaA boxes. Thus, as in Escherichia coli and Bacillus subtilis, the S. lividans dnaA gene may be autoregulated. SPR analysis showed that the affinity of the DnaA protein for a DNA fragment containing both DnaA boxes from the dnaA promoter region (KD = 1.25 nM) is 10 times higher than its affinity for the single 'strong' DnaA box (KD = 12.0 nM). The mobility-shift assay suggests the presence of at least two classes of complex containing different numbers of bound DnaA molecules. The above data reveal that the DnaA protein binds to the two DnaA boxes in a cooperative manner. To deduce structural features of the Streptomyces domain II of DnaA protein, the amino acid DnaA sequences of three Streptomyces species were compared. However, according to the secondary structure prediction, Streptomyces domain II does not contain any common relevant secondary structural element(s). It can be assumed that domain II of DnaA protein can play a role as a flexible protein spacer between the N-terminal domain I and the highly conserved C-terminal part of DnaA protein containing ATP-binding domain III and DNA-binding domain IV.
Majka
J
J
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.
Jakimowicz
D
D
Messer
W
W
Schrempf
H
H
Lisowski
M
M
Zakrzewska-Czerwińska
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015374:Biosensing Techniques; D015153:Blotting, Western; D018983:DNA Footprinting; D004271:DNA, Fungal / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria