Recognition of DNA by omega protein from the broad-host range Streptococcus pyogenes plasmid pSM19035: analysis of binding to operator DNA with one to four heptad repeats.
pSM19035-encoded omega protein forms a dimer (omega2) that binds to a set of 7-bp repeats with sequence 5'-NATCACN-3'. Upon binding to its cognate sites, omega2 regulates transcription of genes required for copy number control and stable inheritance of plasmids, and promotes accurate plasmid segregation. Protein omega2 binds poorly to one heptad but the affinity to DNA increases with two and more unspaced heptads in direct or inverted orientation. DNA titration of increasing numbers of heptads with omega2, monitored by circular dichroism measurements, indicates the binding of one omega2 to one heptad (omega2:heptad stoichiometry of 1:1). Spacing of two directly or inversely oriented heptads by 1 to 7 bp reduces the affinity of the protein for its cognate target site. The binding affinity of omega2 for two directly repeated heptads was severely reduced if one of the base pairs of the core 5'-ATCAC-3' sequence of one of the heptads was individually substituted by any other base pair. Hydroxyl radical footprinting shows a protection pattern at the 5'-ATCAC-3' core. These data suggest that each heptad defines an operator half-site and that tight binding of the symmetric omega2 to the central 5'-TCA-3' core of symmetric or asymmetric targets (differently oriented heptads) is probably achieved by structural changes of DNA and/or protein or both.
de la Hoz
Ana B
AB
Departamento de Biotecnologia Microbiana, Centro Nacional de Biotecnologia, CSIC, 28049 Madrid, Spain.
Pratto
Florencia
F
Misselwitz
Rolf
R
Speck
Christian
C
Weihofen
Wilhelm
W
Welfle
Karin
K
Saenger
Wolfram
W
Welfle
Heinz
H
Alonso
Juan C
JC
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D015964:Gene Expression Regulation, Bacterial; D017665:Hydroxyl Radical / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D013297:Streptococcus pyogenes / Q000235:genetics*; D013816:Thermodynamics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; C060486:methionine repressor protein, Bacteria; D017665:Hydroxyl Radical; D003850:Deoxyribonuclease I