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The transducing phage λh80 dlac carries the lac operator, whereas wild-type λh80 does not. We find that in high salt (0.18 M KCl), ultraviolet radiation causes the formation of a very stable complex between repressor and 5-bromodeoxyuridine (BrdU)-substituted λh80 dlac but not to BrdU-λh80 DNA. Studies with inducers of the lac operon confirm the specificity of attachment. In low slat (0.01 M KCl), ultraviolet radiation will also attach repressor nonspecifically to BrdU-λh80 DNA. The stability of the complex suggests that covalent bonds are formed. We also report that another regulatory protein, the catabolite gene activator protein, can be attached similarly to DNA.

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.

By equilibrium competition experiments, the dissociation constant (K RD ) of lac repressor for E. coli DNA carrying a deletion of the lac operon was measured at a variety of salt concentrations. These data are used in the consideration of several aspects of protein-DNA interaction: •—Quantitative estimates of specificity are made. Specificity changes only slightly with salt concentration. •—We calculate that in vivo, 98% or more of repressor is bound to DNA predominately at sites other than the lac operator. •—Inducers shift repressor from operator to nonoperator DNA, but do not free it from DNA. •—The general affinity of repressor for E. coli DNA is sufficient to support a model where repressor slides along DNA for significant distances. •—The effective dissociation constant of repressor for operator (K eff ) is very sensitive to the total DNA concentration. •—We propose that "junk" DNA in eucaryotes functions to maintain total DNA at an optimum concentration. •—We consider the lac operon in the nucleus of a lymphocyte, point out that severe difficulties would be encountered, and suggest possible solutions.

Using a membrane filter assay, we have obtained results from both kinetic and competition experiments indicating that histones bind more strongly to bromodeoxyuridine-substituted DNA than to normal DNA. At 37 degrees C in our standard buffer of 0.2 M ionic strength, the rate of dissociation of histones H1, H2, and h4 from BrdU-substituted DNA is respectively 7, 4, and 2 times slower than it is from normal DNA. Competition experiments show an even greater difference between BrdU-substituted and normal DNA with respect to histone binding. The tighter binding of histones to BrdU-substituted DNA is of interest because of the known effects of BrdU on eukaryotic chromosome condensation and staining, virus induction, and the inhibition of differentiation.

The interactions 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(U 1 G 1 ) 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 mechanism by which sodium fluoride and cycloheximide inhibit protein synthesis in the rabbit reticulocyte complete, cell-free system has been investigated. Both compounds have been found to inhibit the initiation of new chains on ribosomes. Studies of the rate of amino acid incorporation indicate that cycloheximide also inhibits incorporation into nascent chains that were initiated in intact cells and remain attached to ribosomes during their isolation. Sodium fluoride was found to have no apparent effect on the rate of incorporation into these previously initiated nascent chains, but to cause an apparent breakdown of polysomes to single ribosomes in intact cells. These 80 s ribosomes isolated from sodium fluoride-treated reticulocytes actively incorporate amino acids in the cell-free system and exhibit other properties that indicate they are similar or identical to monomeric ribosomes isolated from untreated cells. These observations are interpreted to indicate a sodium fluoride-sensitive reaction related to the initiation of new peptide chains on ribosomes. The sodium fluoride inhibition of this reaction may bring about the increase in the proportion of monomeric ribosomes because of a failure to re-form polysomes.

Evidence is presented that indicates two reactions that each lead to the ribosome dependent hydrolysis of GTP by two physically distinct transfer enzymes from rabbit reticulocytes. One is dependent on aminoacyl-tRNA and the binding enzyme. The other is associated with TF-II activity and does not require aminoacyl-tRNA. The two enzymes differ in other enzymatic and physical properties.

The highly purified binding enzyme from rabbit reticulocytes is found to function in enzymatic binding of phenylalanyl transfer RNA to ribosomes, ribosome and aminoacyl RNA-dependent hydrolysis of guanosine triphosphate, and as a factor required for the synthesis of peptides. Interdependent reactions are indicated. Guanosine diphosphate and the guanosine triphosphate analogue, 5'-guanylyl methylene diphosphonate, appear to act as competitive inhibitors of guanosine triphosphate in the enzymatic binding reaction. It is concluded that guanosine triphosphate hydrolysis is involved at this step in the sequence of reactions of peptide bond formation. Conversely, guanosine triphosphate hydrolysis is not limited by enzymatic binding, is not dependent upon polyuridylic acid, and appears not to involve a codonanticodon interaction between aminoacyl transfer RNA and messenger RNA. Data are presented that are relevant to the determination of binding enzyme activity in the binding reaction, as a guanosine triphosphatase, and in the synthesis of peptides.

THE lac represser binds very specifically and tightly to the lac operator1–3. In conditions where the repressor binds to operator with a dissociation constant of 10−13 M, the binding of represser to DNA not containing the Escherichia coli lac operator (non-operator DNA) was less tight by several orders of magnitude3. Although weak, we thought this non-operator DNA binding could be highly significant and deserved careful study. We report here on the binding of lac represser to non-operator DNA from several natural sources and also to synthetic poly dAT.

As measured by a decreased rate of dissociation, lac repressor binds 10-times tighter to 5-bromodeoxyuridine-substituted lac operator than it does to normal lac operator. This result is obtained both in the absence and in the presence of isopropylthiogalactoside, an inducing ligand. These data are significant with regard to the mechanism of sequence-specific protein-DNA interaction, and also suggest a possible explanation for the effects of bromodeoxyuridine on the expression of differentiated functions in eukaryotic cells.

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.

Blunt end ligation with an EcoRI/ octanucleotide “linker” was used to construct a plasmid containing chemically synthesized lac operator DNA. This plasmid renders the host bacterium constitutive for β-galactosidase.

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.

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.

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 DNA's 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.

Competition experiments have been used to measure the interaction of Escherichia coli lac represser with many natural and synthetic DNA's 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 K rd , the apparent equilibrium dissociation constant for represser binding to non-operator DNA. The methods are compared and we find that both yield similar K rd values. Earlier work is confirmed that represser binds preferentially to natural DNA's 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 DNA's, 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 DNA's, K rd can be calculated only in weight units, but for poly[d(A-T)]we find that K rd = 1–3 × 10−8 m . The effect of reaction conditions (ionic strength, pH, temperature) on K rd 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.