Dynamics of interaction of RNA polymerase II with nucleosomes. II. During read-through and elongation.
The sulfhydryl-specific fluorescence probe 1,5-IAEDANS (5-(2-((iodoacetyl)amino)ethyl)amino-naphthalene-1-sulfonic acid) was attached to the single cysteine of H3, and reconstituted fluorescent mononucleosomes were used as the template for in vitro transcription by the yeast RNA polymerase II (pol II). DNase I digestion analysis revealed that transcription of nucleosomes by pol II resulted in an overall loosening of the structure. Monitoring the transcription event by steady-state fluorescence analysis showed that nucleosomes only partially open during transcription. This opening is transient in nature, and nucleosomes close back as soon as the pol II falls off the template. Thus, using the technique of fluorescence spectroscopy, partial opening of nucleosome structure could be differentiated from complete dissociation into free DNA and histone octamer, a distinction that may not be possible by techniques like gel electrophoresis. Time-resolved fluorescence emission spectroscopy suggested that during read-through of the template by the pol II, histone octamers do not fall off the DNA. Only minor conformational changes within the histone octamer take place to accommodate the transcribing polymerase.
Bhargava
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P
Centre for Cellular and Molecular Biology, Hyderabad, India.
D000818:Animals; D003850:Deoxyribonuclease I / Q000378:metabolism; D005456:Fluorescent Dyes; D006657:Histones / Q000378:metabolism; D008099:Liver / Q000737:chemistry; D008961:Models, Structural; D009282:Naphthalenesulfonates; D009707:Nucleosomes / Q000378:metabolism*; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents; D013997:Time Factors; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D006657:Histones; D009282:Naphthalenesulfonates; D009707:Nucleosomes; D013439:Sulfhydryl Reagents; C006421:1,5-I-AEDANS; D012319:RNA Polymerase II; D003850:Deoxyribonuclease I