Restriction enzyme accessibility and RNA polymerase localization on transcriptionally active SV40 minichromosomes isolated late in infection

Abstract

The transcriptionally active SV40 minichromosomes isolated late in infection contain a nucleosome-free ORI region or gap. To analyze the chromatin structure of this subpopulation of minichromosomes extracted at different ionic strenghts in the early and late coding regions, minichromosomes were isolated in the presence of a 5, 50, or 130 m M concentration of monovalent cations and subjected to in vitro RNA elongation in either the presence or the absence of high salt and anionic detergent. The minichromosomes isolated at low ionic strength were transcriptionally more active than those isolated at physiological ionic strength. Nevertheless, in each case, the in vitro elongation complexes were present essentially on the late strand of the SV40 genome and localized preferentially in the late and 3′ early coding regions. These regions were transcribed similarly in either the presence or the absence of chromatin denaturing agents. In contrast, the in vitro elongation activity of the RNA polymerase molecules present on the late strand in the middle and 5′ end of the early coding region was inhibited in the absence of treatments to disrupt chromatin structure. In addition, as probed by restriction enzyme digestion, the ORI and late coding regions of the transcriptionally active minichromosomes were found to be more sensitive than the 5′ region of the early genes. Taken together, these results suggest that the 5′ and middle regions of the early genes of the SV40 transcriptional complexes isolated late in infection at low or physiological ionic strenght are packaged in a more compact conformation than the rest of the genome.