Relevance of individual Mo-box nucleotides to DNA binding by the related molybdenum-responsive regulators MopA and MopB in Rhodobacter capsulatus.
Either of two related molybdenum-responsive regulators, MopA and MopB, of Rhodobacter capsulatus is sufficient to repress the nitrogen-fixation gene anfA. In contrast, MopA (but not MopB) activates mop, which codes for a molybdate (Mo)-binding molbindin. Both regulators bind to conserved cis-regulatory elements called Mo-boxes. Single-base substitution of two highly conserved nucleotides within the anfA-Mo-box (T21C and C24T) had little effect on regulator binding and anfA expression as shown by DNA mobility shift assays and reporter gene fusions, respectively. In contrast to C24T, mutation C24A strongly diminished binding and repression by MopA and MopB, showing that different nucleotide substitutions at the same position may have very different effects. A triple mutation destroying the left half-site of the mop-Mo-box completely abolished mop expression by MopA, demonstrating the importance of the mop-Mo-box for mop activation. Two point mutations (T23A and T24C) still allowed binding by MopA, but abolished mop activation, most likely because these nucleotides overlap with the RNA polymerase-binding site. A mutant mop promoter, in which the mop-Mo-box was exchanged against the anfA-Mo-box, allowed activation by MopA, showing that a former repressor-binding site may act as an activator-binding site depending on its location relative to the other promoter elements.
Müller
Alexandra
A
Lehrstuhl für Biologie der Mikroorganismen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, Bochum, Germany.
Püttmann
Lucia
L
Barthel
Romy
R
Schön
Matthias
M
Lackmann
Jan-Wilm
JW
Narberhaus
Franz
F
Masepohl
Bernd
B
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D055029:Inverted Repeat Sequences; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; C081166:mopA protein, Rhodobacter capsulatus; C081167:mopB protein, Rhodobacter capsulatus; C044659:molybdate; D008982:Molybdenum; D004247:DNA