ARTIFICIAL NUCLEOSOME POSITIONING SEQUENCES TESTED IN YEAST MINICHROMOSOMES - A STRONG ROTATIONAL SETTING IS NOT SUFFICIENT TO POSITION NUCLEOSOMES INVIVO

DNA sequences that support bending around the histone octamer ('rotational setting') are considered to be a major determinant of nucleosome positions. TG5 is an artificial positioning sequence containing 100 bp of an (A/T)3NN(G/C)3NN motif repeated with a 10 bp period. It provides a strong rotational setting and is superior to natural sequences in nucleosome formation in vitro [Shrader, T.E. and Crothers, D.M. (1989) Proc. Natl. Acad. Sci. USA, 86, 7418-7422]. To investigate the contribution of the rotational setting to nucleosome positioning in vivo, TG sequences were inserted in a nucleosome, at the edge of a nucleosome and in a nuclease sensitive region of yeast minichromosomes and the chromatin structures were analysed. In none of the constructs were TG sequences folded in a positioned nucleosome, demonstrating that the rotational setting played a subordinate role in the rough positioning in vivo. The rotational setting might fine tune the positions. Positioned nucleosomes were found overlapping the ends of TG, indicating that a discontinuity of the 10 bp periodicity of (A/T)3 and (G/C)3 near the centre of a nucleosome might be favourable for positioning and serve as a translational signal.