Tetrameric lac repressor may bind to two lac operators on one DNA fragment and induce the intervening DNA to form a loop. Electron microscopy, non-denaturing polyacrylamide gel electrophoresis, and DNase I protection experiments were used to demonstrate such DNA loops, where the distance between the centres of symmetry of the two lac operators varies between 63 and 535 bp. Formation of a DNA loop is favoured by correct phasing of the two lac operators and a low concentration of both components of the reaction. When a large excess of lac repressor over DNA is used, a 'tandem' structure is observed, in which both lac operators are occupied independently by two repressor tetramers. When the concentrations of both lac repressor and lac operator are high, a 'sandwich' structure is observed, in which two DNA molecules are connected by two lac repressor tetramers in trans.

We have studied the ability of the histone (H3-H4)2 tetramer, the central part of the nucleosome of eukaryotic chromatin, to form particles on DNA minicircles of negative and positive superhelicities, and the effect of relaxing these particles with topoisomerase I. The results show that even modest positive torsional stress from the DNA, and in particular that generated by DNA thermal fluctuations, can trigger a major, reversible change in the conformation of the particle. Neither a large excess of naked DNA, nor a crosslink between the two 113s prevented the transition from one form to the other. This suggested that during the transition, the histones neither dissociated from the DNA nor were even significantly reshuffled. Moreover, the particles reconstituted on negatively and positively supercoiled minicircles look similar under electron microscopy. These data agree best with a transition involving a switch of the wrapped DNA from a leftto a right-handed superhelix. It is further proposed, based on the left-handed overall superhelical conformation of the tetramer within the octamer [Arents, G., Burlingame Nucleosome dynamics is a necessary requirement of DNA function in chromatin, including transcription, replication, or repair. It has long been thought to be mediated by the tripartite organization of the histone octamer, made of an (H3-H4)2 tetramer bound with two H2A-H2B dimers (1). Thermodynamic studies of octamer assembly and disassembly showed that the forces holding the tetramer and dimers together are of a different nature and much stronger than the forces binding the dimers to the tetramer (1, 2), inspite of an extensive dimer-tetramer interface. This interface is disrupted in the first step of octamer disassembly.The crystal structure of the nucleosome core particle (3), and even more so, of the histone octamer (4), subsequently confirmed this tripartite organization. Moreover, one of the H2A-H2B dimers in the crystal structure of the core particle appears significantly displaced from its original position (3), which probably reflects the effects of crystal-packing forces and the relative ease with which the dimers can move relative to the tetramer. Such dimer lability may also be important in vivo, as is emphasized by the observations of an H2A-H2B deficit in nucleosome cores originating from transcriptionally active chromatin (5), and of H2A-H2B exchange with the endogenous histone pool upon chromatin transcription (6-8).Here we provide evidence for a potential conformational flexibility of the (H3-H4)2 tetramer, a tribute to the role of this tripartite organization of the histone octamer in nucleosome dynamics. This conformational flexibility is suggested by the observation of a similar affinity of the tetramer for negatively and positively supercoiled DNA minicircles, which apparently arises from the ability of the wrapped DNA to switch from a left-to a right-handed superhelix. This transition is found to require only modest positive torsional stress in the DNA, and can be triggered by DN...

Transcription factors that are bound specifically to DNA often interact with each other over thousands of base pairs [1] [2]. Large DNA loops resulting from such interactions have been observed in Escherichia coli with the transcription factors deoR [3] and NtrC [4], but such interactions are not, as yet, well understood. We propose that unique protein complexes, that are not present in solution, may form specifically on DNA. Their uniqueness would make it possible for them to interact tightly and specifically with each other. We used the repressor and operators of coliphage lambda to construct a model system in which to test our proposition. lambda repressor is a dimer at physiological concentrations, but forms tetramers and octamers at a hundredfold higher concentration. We predict that two lambda repressor dimers form a tetramer in vitro when bound to two lambda operators spaced 24 bp apart and that two such tetramers interact to form an octamer. We examined, in vitro, relaxed circular plasmid DNA in which such operator pairs were separated by 2,850 bp and 2,470 bp. Of these molecules, 29% formed loops as seen by electron microscopy (EM). The loop increased the tightness of binding of lambda repressor to lambda operator. Consequently, repression of the lambda PR promoter in vivo was increased fourfold by the presence of a second pair of lambda operators, separated by a distance of 3,600 bp.

The Bacillus subtilis LrpC protein belongs to the Lrp/ AsnC family of transcriptional regulators. It binds the upstream region of the lrpC gene and autoregulates its expression. In this study, we have dissected the mechanisms that govern the interaction of LrpC with DNA by electrophoretic mobility shift assay, electron microscopy, and atomic force microscopy. LrpC is a structurespecific DNA binding protein that forms stable complexes with curved sequences containing phased A tracts and wraps DNA to form spherical, nucleosomelike structures. Formation of such wraps, initiated by cooperative binding of LrpC to DNA, results from optimal protein/protein interactions specified by the DNA conformation. In addition, we have demonstrated that LrpC constrains positive supercoils by wrapping the DNA in a right-handed superhelix, as visualized by electron microscopy.

A map of local curvature of the pBR322 DNA has been established by electron microscopy analysis of linearized plasmid molecules. To determine their polarity these molecules are one end labelled with an avidin‐ferritin‐biotin complex and the images are digitized. Local curvature is calculated from two mathematical treatments of the DNA trajectory and expressed in term of a mean dinucleotide wedge angle. Eight regions of curvature are distinguished. The four main regions of curvature have a high content of phased AA runs. The experimental curvature map is compared to theoretical maps of curvature obtained from four available models for DNA curvature.

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