Several polynucleotides that assume an Aform helical structure in solution are unable to form nucleosomes. We attempted to establish a relationship between the ease of the A-form -+ B-form helix transition and ease of nucleosome formation by reconstituting nucleosomes using ribosubstituted DNA containing various levels of ribonucleotides. Instead we discovered that, when riboadenosine is substituted for deoxyriboadenosine, even one ribonucleotide per 125 base pairs of DNA reduces nucleosome formation and that DNA containing >5% ribonucleotide is completely unable to form nucleosomes. Ribosubstituted DNA restriction fragments exhibited altered mobility on native 6% polyacrylamide gels, indicating an altered helical structure (probably bending).The effects on both nucleosome formation and gel mobility are nucleotide specific and are correlated, being greatest for riboadenosine and decreasing in the order riboadenosine > riboguanosine > ribocytosine. The results are consistent with the hypothesis that the rate of nucleosome formation can be drastically reduced by isolated local perturbations, such as kinking or bending, in the helical structure of DNA.We are interested in the manner by which DNA helix structure modulates its own mode of packaging by histones in chromatin. In some cases the effects on packaging have obvious physiological implications. For example, the positioning of artificial nucleosomes on Escherichia coli lac operator DNA (1) constrains the DNA in such a way that the repressor binding surface of the DNA always faces outward to bind repressor (2). Eukaryotic examples include the specific positioning of nucleosomes on 5S gene DNA (3) so as to facilitate binding by transcription factor IIIA (4) and the avoidance by nucleosomes of poly(dA)-poly(dT) tracts in DNA (5, 6), which probably accounts for the promoteractivating properties of this sequence in chromatin (7).It now appears that the sequence-specific positioning of nucleosomes and the sidedness with which DNA binds the histone core may be explained by the tendency of certain short sequence elements (8) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.