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. 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.
Chirinos
M
M
Centro de Biología Molecular "Severo Ochoa,", Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, Madrid, 28049, Spain.
Hernández
F
F
Palacián
E
E
D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D002645:Chickens; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005814:Genes, Viral; D006657:Histones / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020836:Protein Structure, Quaternary; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins
D004274:DNA, Recombinant; D004279:DNA, Viral; D006657:Histones; D046911:Macromolecular Substances; D009707:Nucleosomes; D014764:Viral Proteins; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases