Faculty Bibliography

Accurate and quantitative transcription of the tk gene requires sequence elements that reside within 110 nucleotides of 5'-flanking DNA. We have determined the boundaries of a hypersensitive region in 5'DNA flanking the tk gene by analyzing the relative sensitivity of specific restriction sites clustered in this region. Five different restriction enzymes, recognizing over 50 sites in the promoter region of the tk gene, each show a preferential cleavage in nuclei at a restricted number of sites residing between positions -4 and -182. Thus the tk promoter sequences are contained entirely within a hypersensitive region. Analysis of this site in tk+ transformants, a tk- mutant and tk+ rerevertant indicates that expression of tk RNA is correlated with structural alterations in the tk promoter

We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli

We have shown that the lac repressor can recognize and bind specifically to the lac operator contained in short restriction fragments which have been complexed with the four core histones to form artificial nucleosomes and core particles. These lac reconstitutes have been well characterized, and it is apparent that the operator DNA itself is associated fully and normally with the octameric histone cores. The binding of repressor to these reconstitutes is operator dependent since nucleosomes lacking the operator sequence fail completely to bind repressor under our conditions. Moreover, binding is abolished by IPTG (isopropyl thiogalactoside), further demonstrating operator specificity. Nevertheless, sedimentation studies show that repressor binding does not involve displacement of the histone octamer. Thus, the lac repressor and the histone octamer bind simultaneously to the same DNA. lac reconstitutes, in which the DNA has been cross-linked to the histones with formaldehyde, also support simultaneous specific binding by lac repressor. Since all particles among the reconstitutes, cross-linked or not, bind repressor quantitatively, we infer that the repressor binding surface of the operator DNA always faces generally outward rather than inward toward the histone core. It is likely to be this feature of lac operator particle structure, dictated in an unknown manner by DNA sequence, that allows the simultaneous binding of histones and repressor to the same DNA region

Restriction fragments, 203 and 144 base pairs in length, bearing the Escherichia coli lac control region have been reconstituted with the core histones from calf thymus to form nucleosomes. By several criteria the reconstituted nucleosomes are similar to native nucleosomes obtained by micrococcal nuclease digestion of calf thymus nuclei. However, sensitive nuclease digestion studies reveal subtle and important differences between native monosomes and the lac reconstitutes. Each reconstitute consists mainly of nucleosomes containing histone cores placed nonrandomly with respect to the DNA sequence. The shorter reconstitute forms asymmetric nucleosomes as evidenced by the DNase I digestion pattern. Exonuclease III digestion followed by 5'-end analysis of the larger reconstitute suggests that, of the many possible arrangements of histone core with DNA sequence, only two are highly favored