Cooperative and Anticooperative Effects in Binding of the First and Second Plasmid OsymOperators to a LacI Tetramer: Evidence for Contributions of Non-operator DNA Binding by Wrapping and Looping

Thermodynamics of the interactions of Lac repressor with variants of the symmetric Lac operator: effects of converting a consensus site to a non-specific site

“…This behavior is exempli®ed by Figure 3(b) which plots log 10 K sym as a function of 1/T; K sym exhibits a maximum at $290 K. Analysis of these data (see equation (7) ), a common feature of site-speci®c protein-nucleic acid interactions Spolar & Record, 1994 Levandoski, 1995)? The amount of non-polar surface buried in the LacI-O sym contact interface (Chuprina et al, 1993;Lewis et al, 1996) is far less than the amount predicted from a quantitative analysis of ÁC o obs in terms of changes in water-accessible surface area Spolar et al, 1989;Spolar & Record, 1994).…”

“…SK obs for the binding of LacI to wild-type operator O 1 also is smaller in magnitude for a 40 bp DNA than for a 4600 bp DNA (Whitson & Matthews, 1986). We have recently proposed that the observed increase in jSK obs j with DNA length arises from changes in the nature of LacI-operator complexes as the length of non-operator DNA¯anking the operator site increases (Levandoski et al, 1996). For lac operators embedded in fragments longer than 40 bp, we hypothesize that at low [salt],¯anking non-operator sequences interact with LacI nonspeci®cally via local wrapping and that on suf®-ciently long fragments (plasmid-length), distant non-operator DNA sites bind at the second site via looping.…”

“…LacI-DNA interactions serve as a key paradigm for control of genetic expression, providing a model system for characterizing the relationship between DNA site recognition and transcription regulation (cf Capp et al, 1996;Levandoski et al, 1996;Lewis et al, 1996;Matthews, 1996;Mu È ller et al, 1996, and references therein). A de®ning characteristic of LacI is its speci®city of discrimination between operator and non-operator DNA sequences: depending on solution conditions, the equilibrium association constant for the wild-type operator sequence (O 1 ) exceeds that for any non-speci®c site by at least six to seven orders of magnitude (Lin & Riggs, 1975;Record et al, 1977).…”

“…Thus, from the available structural data, we estimate that a net of six positive charges on two LacI headpieces contact phosphate oxygen atoms in the vicinity of one O sym operator. Binding to double-stranded DNA of an oligolysine (or other oligocation) with charge Z 6 exhibits an SK obs of $ À 5.4 at a similar [salt] (Record et al, 1976;Mascotti & Lohman, 1993) (Ha et al, 1992;Levandoski, 1995); for a 107 bp DNA, SK sym À 6.6 AE 0.8 (Capp et al, 1996). SK obs for the binding of LacI to wild-type operator O 1 also is smaller in magnitude for a 40 bp DNA than for a 4600 bp DNA (Whitson & Matthews, 1986).…”

Proteins mediating DNA loops effectively block transcription

“…of incubation (see Materials and Methods), LacI has established a dynamic equilibrium with operator sites along DNA molecules. However, 7.5 nM LacI fairly saturates Os operators ( K d  < 1 × 10 −14 M in similar K + conditions29), and the estimated tether‐augmented, effective concentration ( J value) of LacI is expected to be in the range of 4 to 100 nM for a 400 bp loop (data not shown30). Therefore, operator occupancy is expected to be very high throughout the 60 s incubation.…”