Binding of MetJ repressor to specific and nonspecific DNA and effect of S-adenosylmethionine on these interactions - PubMed (original) (raw)

Binding of MetJ repressor to specific and nonspecific DNA and effect of S-adenosylmethionine on these interactions

Anne M Augustus et al. Biochemistry. 2010.

Abstract

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.

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Figures

Figure 1

Gel shift showing MetJ-R binding to various DNA oligos in the absence (A) or presence (B) of 1 mM SAM, and in the presence of SAM and excess MetJ (C). The DNA is stained with a dye that fluoresces green while MetJ-R fluoresces red. The molecular weight marker is Hyper Ladder V from Bioline.

Figure 2

Sedimentation equilibrium study of MetJ-R alone (A) and in complex with 5con DNA (B). Residuals are in units of percent.

Figure 3

MWb values for MetJ-R complexes with DNA containing zero (NS44) or one metbox (NS1con) at various MetJ:DNA ratios. MWb for both DNAs is ~12,040. The theoretical MWb for a 1:1 MetJ:DNA complex is ~18,200 and for a 2:1 complex is ~24,400.

Figure 4

MWb values for MetJ-R complexes with DNA containing two metboxes (2con and 2nat). MWb for both DNAs is ~5440. The theoretical MWb for a 1:1 MetJ:DNA complex is ~11,600 and for a 2:1 complex is ~17,800.

Figure 5

MWb values for MetJ-R complexes with DNA containing five metboxes (5con and 5nat). MWb for both DNAs is ~12040. The theoretical MWb for a 1:1 MetJ:DNA complex is ~18,200, for a 2:1 complex is ~24,400, for a 3:1 complex is ~30,600, for a 4:1 complex is ~36,700 and for a 5:1 complex is ~42,900. The upper dashed line represents the 5:1 complex, while the lower dashed line indicates the re-equilibration curve expected for a non-cooperative process.

Figure 6

MWb values for complexes containing MetJ-R and DNA with five metboxes as a function of SAM concentration. The DNAs used were 5nat (Δ, MWb =12038), and 5con (●, MWb =12040). The DNA:MetJ was always at a 1:1 [metbox]/[MetJ-R] ratio. Stoichiometric SAM is 20 µM, indicated by the vertical dashed line between 10 and 100 µM SAM.

Figure 7

Average number (n) of MetJ-R dimers bound to stoichiometric concentrations of various DNAs in the presence (filled symbols) or absence (open symbols) of 1 mM SAM. The value of n was determined from the data using Equation 2.

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Binding of MetJ repressor to specific and nonspecific DNA and effect of S-adenosylmethionine on these interactions - PubMed (original) (raw)