DNA AND PROTEIN DETERMINANTS OF NUCLEOSOME POSITIONING ON SEA-URCHIN 5S RIBOSOMAL-RNA GENE-SEQUENCES INVITRO

DNA and protein determinants of nucleosome positioning have been examined after in vitro reconstitutions of native or modified histone octamers onto tandem repeats of 207- and 172-base-pair DNA sequences containing the Lytechinus variegatus 5S rRNA gene and onto monomeric sequences derived from these by digestion with various restriction endonucleases. In all cases, a major nucleosome position as well as a number of minor positions have been observed, which indicates that the generation of multiple positions is an inherent property of the 5S rRNA gene sequence. Interestingly, all positions observed differ by multiples of 10 base pairs. Data obtained under different reconstitution conditions demonstrate that the observed distributions of nucleosomes on these DNA templates are equilibrium distributions. This study has also examined the positioning of histone octamers from which histone 'tails' had been removed by tryptic digestion. Results indicate that the histone tails are not determinants of nucleosome positioning. Although our results suggest that the mechanical properties of the 5S rDNA are the fundamental factors determining nucleosome positioning, they are insufficient to direct all nucleosomes into a single location.