Structure-function relationships in nucleosomal arrays containing linker histone H5.

To study the structural and functional changes accompanying the integration of histone H5 into the nucleosome structure, linear DNA species have been employed with a terminal promoter for bacteriophage T7 RNA polymerase followed by tandem repeats of a 207-bp nucleosome positioning sequence. The oligonucleosomes assembled from 12-repeat DNA and saturating amounts of core histone octamer plus histone H5 are compacted, in the presence of 1 mM free magnesium ions, to the level of the 30-nm fiber. Under these ionic conditions the efficiency in RNA synthesis and the size distribution of RNA chains obtained with this template are the same as those corresponding to the template without H5, indicating that the 30-nm fiber stabilized by H5 does not impair RNA elongation.

Transcription of DNA templates associated with histone (H3 x H4)(2) tetramers.

To investigate the in vitro transcription by bacteriophage T7 RNA polymerase of oligonucleosomes lacking histone H2A x H2B dimers, templates were assembled from histone (H3 x H4)(2) tetramers with and without the complementary amount of H2A x H2B dimers and two different DNA species: pGEMEX-1, devoid of nucleosome positioning sequences, and T7-207-18, which contains downstream from the promoter 18 tandem repeats of a 207-bp positioning sequence. Assembly with core histone octamers affects pGEMEX-1 transcription mainly at the initiation level, while T7-207-18 is almost exclusively inhibited at the level of elongation. With both DNA templates and under different salt conditions, RNA synthesis is much more efficient on oligonucleosomes containing only (H3 x H4)(2) tetramers than on those with whole histone octamers.

Transcriptional inhibitory role of the tail domains of histone (H3 x H4)2 tetramers.

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from a plasmid containing a promoter and a terminator for this polymerase, (H3 x H4)2 tetramers deprived of their tail domains, and H2A x H2B dimers. Histone (H3 x H4)2 tetramers lacking their terminal domains were obtained from trypsin-digested nucleosomal cores. The oligonucleosomal templates containing (H3 x H4)2 tetramers lacking their tail domains, like the control templates with intact core histone octamers, protect approximately 146 base pairs of DNA against micrococcal nuclease digestion.

Repressive effect on oligonucleosome transcription of the core histone tail domains.

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from histone octamers and three different DNA species, two circular (pGEMEX-1 and pT207-18) and one linear (T7-207-18). pGEMEX is devoid of nucleosome positioning sequences, while in pT207-18 and T7-207-18 the region downstream of the promoter contains 18 tandem repeats of a 207 bp positioning sequence derived from the 5S RNA gene of the sea urchin Lytechinus variegatus. Elimination of the histone tails in the assembled oligonucleosomes by trypsin digestion is accompanied, in all three DNA species, by substantial increases in transcription efficiency, assayed at different KCl and MgCl2 concentrations, after allowing for the aggregation observed under certain conditions.

Transcriptional properties of oligonucleosomal templates containing acetylated (H3-H4)2 tetramers.

Direct chemical acetylation of an oligonucleosomal template for bacteriophage T7 RNA polymerase is accompanied by a substantial increase in its capability to support RNA synthesis. The template was assembled from a plasmid, containing a promoter and a terminator for T7 RNA polymerase, plus one (H3-H4)2 tetramer and two H2A.H2B dimers for each 200 base pairs of DNA.