Transcription of DNA Templates Associated with Histone (H3 · H4)2 Tetramers

Abstract

To investigate the in vitro transcription by bacteriophage T7 RNA polymerase of oligonucleosomes lacking histone H2A · H2B dimers, templates were assembled from histone (H3 · H4)2 tetramers with and without the complementary amount of H2A · 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 · H4)2 tetramers than on those with whole histone octamers. Under conditions promoting a low transcription rate, it is unambiguously shown with pGEMEX-1 that the block to initiation due to the presence of core histone octamers is substantially removed when (H3 · H4)2 is substituted for the whole octamer. With T7-207-18, under assay conditions allowing transcription of the whole coding region of the naked DNA, analysis of the transcription products indicates that RNA elongation on the template containing (H3 · H4)2 tetramers takes place as easily as on free DNA, in contrast with the significant inhibition observed in the presence of whole histone octamers.