Binding of MetJ repressor to specific and nonspecific DNA and effect of S-adenosylmethionine on these interactions.
We have used analytical ultracentrifugation to characterize the binding of the methionine repressor protein, MetJ, to synthetic oligonucleotides containing zero to five specific recognition sites, called metboxes. For all lengths of DNA studied, MetJ binds more tightly to repeats of the consensus sequence than to naturally occurring metboxes, which exhibit a variable number of deviations from the consensus. Strong cooperative binding occurs only in the presence of two or more tandem metboxes, which facilitate protein-protein contacts between adjacent MetJ dimers, but weak affinity is detected even with DNA containing zero or one metbox. The affinity of MetJ for all of the DNA sequences studied is enhanced by the addition of SAM, the known cofactor for MetJ in the cell. This effect extends to oligos containing zero or one metbox, both of which bind two MetJ dimers. In the presence of a large excess concentration of metbox DNA, the effect of cooperativity is to favor populations of DNA oligos bound by two or more MetJ dimers rather than a stochastic redistribution of the repressor onto all available metboxes. These results illustrate the dynamic range of binding affinity and repressor assembly that MetJ can exhibit with DNA and the effect of the corepressor SAM on binding to both specific and nonspecific DNA.
Augustus
Anne M
AM
Department of Biochemistry, Duke University, Durham, North Carolina 27710, USA.
Sage
Harvey
H
Spicer
Leonard D
LD
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D031521:Fractionation, Field Flow; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000494:pharmacology*; D014461:Ultracentrifugation / Q000379:methods
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; C060486:methionine repressor protein, Bacteria; D012436:S-Adenosylmethionine; D004247:DNA; D008715:Methionine