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

• Published in: Archives of biochemistry and biophysics 1999-10, Vol.370 (2), p.222-230
• Main Authors: Chirinos, M, Hernández, F, Palacián, E
• Format: Article
• Language: English
• Subjects: Animals; Bacteriophage T7 - enzymology; Bacteriophage T7 - genetics; Chickens; DNA - genetics; DNA - metabolism; DNA, Recombinant - genetics; DNA, Recombinant - metabolism; DNA, Viral - genetics; DNA, Viral - metabolism; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Genes, Viral; Histones - chemistry; Histones - metabolism; In Vitro Techniques; Macromolecular Substances; Molecular Weight; Nucleosomes - chemistry; Nucleosomes - metabolism; Promoter Regions, Genetic; Protein Structure, Quaternary; RNA - biosynthesis; RNA - genetics; Transcription, Genetic; Viral Proteins
• ISSN: 0003-9861
• DOI: 10.1006/abbi.1999.1392

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. 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 x 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 x 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.