Mycobacterium tuberculosis DnaA initiator protein: purification and DNA-binding requirements.
The Mycobacterium tuberculosis oriC (the origin of chromosomal replication) region contains 13 non-perfect DnaA boxes. The M. tuberculosis initiator protein, DnaA, was overexpressed in Escherichia coli as a soluble His-tagged fusion protein. The purified protein His6MtDnaA was investigated for its binding properties to DnaA boxes from the oriC region. Gel retardation demonstrated that the DnaA from M. tuberculosis requires two DnaA boxes for efficient binding. Electron microscopy as well as DNase I footprinting showed that the His6MtDnaA protein binds to four specific regions, which correspond to the location of 11 out of 13 previously identified DnaA boxes within the M. tuberculosis oriC. Probably, in M. tuberculosis, DnaA molecules by co-operative binding of numerous 'non-perfect' DnaA boxes assemble along the oriC region and subsequently form a massive nucleoprotein complex.
Zawilak
Anna
A
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. Weigla 12, 53-114 Wrocław, Poland.
Kois
Agnieszka
A
Konopa
Grazyna
G
Smulczyk-Krawczyszyn
Aleksandra
A
Zakrzewska-Czerwińska
Jolanta
J
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D011485:Protein Binding; D018741:Replication Origin / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria