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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.

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 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.

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.