Syr-Yaung Lin

lac represser binding to non-operator DNA: Detailed studies and a comparison of equilibrium and rate competition methods☆

Competition experiments have been used to measure the interaction of Escherichia coli lac represser with many natural and synthetic DNAs that do not contain the lac operator (non-operator DNA). Two types of experiments were done: (1) equilibrium competition experiments, where the effect of competing DNA on the equilibrium concentration of represser-operator complex was measured, and (2) rate competition experiments, where the effect of competing DNA on the rate of represser-operator complex formation was measured. The second method is an order of magnitude more sensitive than the first. For both types of experiments, we present methods for the estimation of Krd, the apparent equilibrium dissociation constant for represser binding to non-operator DNA. The methods are compared and we find that both yield similar Krd values. Earlier work is confirmed that represser binds preferentially to natural DNAs of high A + T content and that poly[d(A–T)]is a very good competitor. Poly[r(A,U)], tRNA, and rRNA are very poor competitors. The glucosylated DNAs, T2 and T4, compete well, about as expected from their A + T content. Denatured non-operator DNA competes approximately the same as native DNA. Effector ligands could not be shown to affect non-operator binding. For most DNAs, Krd can be calculated only in weight units, but for poly[d(A-T)]we find that Krd = 1–3 × 10−8 m. The effect of reaction conditions (ionic strength, pH, temperature) on Krd was investigated. Contrary to earlier conclusions, non-operator DNA binding is not preferentially reduced at high ionic strength. These results are significant for several areas of current interest: (a) the fractionation and purification of other DNA-binding proteins, (b) the general problem of demonstrating specific DNA binding, and (c) the deatiled mechanism of regulatory protein-DNA interaction.

Lac repressor binding to operator analogues: Comparison of poly[d(A-T)], poly[d(A-BrU)], and poly[d(A-U)]

Abstract Previous work has established that Escherichia coli lac repressor binds strongly to poly[d(A-T)]. Here we use competition experiments to measure repressor binding to synthetic DNAs related to this polymer. Poly[d(A-BrU)] competes about forty times more effectively for repressor than does poly[d(A-T)], whereas poly[d(A-U)] competes twenty fold less effectively. This result suggests that the atom or group occupying the 5-position of the pyrimidine ring is important for recognition by repressor.

The binding of lac repressor and the catabolite gene activator protein to halogen-substituted analogues of poly[d(A-T)].

We have measured the binding of two regulatory proteins to the complete halogen-substituted series of poly[d(A-T)] analogues. Both the lac repressor and the catabolite gene activator protein were found to bind more strongly to all of the halogen-substituted DNAs than they do to poly[d(A-T)]. For both proteins, the order of binding preference is poly[d(A-ioU)] is greater than poly[d(A-brU)] is greater than or equal to poly[d(A-clU)] is greater than poly[d(A-flU)] is greater than poly[d(A-T)]. Quantitative data on the binding of these proteins to poly[d(A-U)] is also given. The significance of these results for the mechanism of protein-DNA interaction is discussed. This is the first report that an activator protein binds more strongly to a halogen-substituted DNA; we discuss this result with regard to the mechanism of action of bromodeoxyruidine and other halogen-substituted base analogues on the inhibition and induction of differentiation in eucaryotic cells.

A comparison of lac repressor binding to operator and to nonoperator DNA.

Abstract We have compared the operator and nonoperator DNA binding activities of the lac repressor with respect to inactivation or inhibition by trypsin, heat, actinomycin, and isopropylthiogal-actoside. The two DNA binding activities were found to differ only in their sensitivity to the inducing ligand isopropylthiogal-actoside. Repressor binding to poly(dT-dT-dG)·poly(dC-dA-dA) was shown not to be affected by isopropylthiogalactoside.

The binding of androgen receptor to DNA and RNA

The androgen receptor from mouse kidney cytosol has been studied for its nucleic acid binding properties by DNA-cellulose centrifugation assay. The receptor appears to bind to RNA (mRNA, tRNA, rRNA) as well as to DNA. Salt and heat activation of the androgen receptor enhances both DNA and RNA binding. The receptor binds slightly better to denatured DNA than to native DNA. The androgen receptor binds about 2-fold tighter to poly(dG-dC) than to poly (dA-dT). The interaction of the receptor with DNA is not greatly affected by the BrdUrd substitution. The observation that androgen receptor shows a significant affinity to RNA may imply that androgen receptor-RNA interaction could play a role in gene regulation.

Interactions of nuclear estrogen receptor with DNA and RNA

The interaction of the nuclear estrogen receptor from hen oviduct with nucleic acids were studied by competition assay using DNa-cellulose centrifugation. We demonstrated that the estradiol-receptor complex binds similarly well to poly(A) RNA and denatured DNA. The estrogen receptor was found to interact more strongly with poly(G), poly(U) than with poly(A), poly(C). The receptor complex binds similarly to poly(A) and poly(dA), and to poly(U) and poly(dU). However, the receptor complex shows stronger binding to poly(G) than to poly(dG) and to poly(C) than to poly(dC). Studies with heteropolyribonucleotides indicated that poly(U1G1) is more effective in competing for the estrogen receptor, and poly(AC) and poly(AUG) are moderately effective, whereas poly(ACU) is least effective. GMP and dGMP showed some competition for the nuclear receptor at 300-fold higher nucleotide concentrations than that of the synthetic poly(G). Observations that the nuclear estrogen receptor binds to poly(A) RNA and interacts selectively with polyribonucleotides suggest that the estrogen receptor-RNA interaction may play a role for the function of estrogens in gene regulation.

THE INTERACTIONS OF BrdU-SUBSTITUTED DNA WITH LAC REPRESSOR AND HISTONES

The interactions of lac repressor and histones to BrdU-substituted DNA and the normal DNA has been studied by nitrocellulose membrane filter methods. Competition with unlabeled operator DNA and poly[d(A-T)] indicates that lac repressor binds about ten fold tighter to BrdU-substituted operator DNA than to the normal operator DNA. Inducer inhibition and heat inactivation studies show that repressor-BrdU operator DNA complex is more stable than the repressor-normal operator complex. The interaction of BrdU-operator DNA to repressor appears to be more sensitive to pH temperature and ionic strength than the normal operator DNA. Histones, H1 and H4 bind more tightly to poly[d(A-BrU)] than to poly[d(A-T)]. The histone interaction with BrdU-DNA is highly susceptible to the presence of urea. This suggests that one reason for the increased affinity of BrdU-DNA to proteins is due to an enhancement of the hydrophobic interaction. The increased affinity of BrdU-DNA to lac repressor and histones may at least partially explain the specific effects of BrdU in gene regulation.

THE INTERACTION OF CHEMICALLY SYNTHESIZED 21 BASE PAIR LAC OPERATOR WITH THE LAC REPRESSOR

ABSTRACT The interaction of lac repressor with 21 base pair lac operator, chemically synthesized by a phosphotriester method, has been studied in considerable detail. The binding of the synthetic operator to the lac repressor is similar to that of natural operator in lambda plac DNA in its response to salt, temperature, and effector ligands. A good response is seen with IPTG, an inducer, and ONPF, an anti-inducer. The affinity of 21 base pair operator for repressor is less than that of natural operator in lambda plac, but the reduction in affinity is close to that predicted from a consideration of the involvement of unspecific interactions with adjacent DNA in the overall binding mechanism for natural operator.