Dynamics of interaction of RNA polymerase II with nucleosomes. I. Effect of salts.
Mononucleosomes were labeled with the sulfhydryl-specific fluorescence probe 1,5-IAEDANS (5-(2-((iodoacetyl)amino)ethyl)amino-naphthalene-1-sulfonic acid) by attaching the dye to the single cysteine of H3 through a covalent linkage. The enzyme RNA polymerase II (pol II) utilized the native and the reconstituted fluorescent nucleosomes as templates with greatest efficiency when 0.2 M potassium acetate (AcOK) was used as the supporting salt; 0.2 M NaCl was found to be very much inhibitory. Measurement of polarity of the microenvironment of the dye at its binding site in the nucleosome showed the conformation to be more open in the presence of AcOK, compared to that in 0.1 or 0.2 M NaCl. The binding of pol II to the nucleosome resulted in a relatively more compact structure when measured in terms of the polarity of the microenvironment of the dye in various salt-dependent conformations of the nucleosomes. Time-resolved fluorescence spectroscopy showed that the probe molecule at its binding site undergoes certain excited-state processes, and the presence/absence or rate of these excited-state processes depends on the conformation of nucleosomes, which in turn depends on the type and concentration of the ion present in the medium. Time-resolved emission spectra showed that binding of nucleosomes by pol II established some new contacts that resulted in inaccessibility of the dye to the bulk solvent, reflecting a more hydrophobic environment for the dye in the steady-state spectra. Thus, binding or transcription of nucleosomes by pol II did not break open their structure. Rather, some transient internal adjustments within the histone octamer may take place to accommodate the bulky pol II molecule.
Bhargava
P
P
Centre for Cellular and Molecular Biology, Hyderabad, India.
D000085:Acetates / Q000494:pharmacology; D019342:Acetic Acid; D000818:Animals; D005456:Fluorescent Dyes; D008099:Liver / Q000737:chemistry; D008956:Models, Chemical; D009282:Naphthalenesulfonates; D009707:Nucleosomes / Q000187:drug effects* / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D012319:RNA Polymerase II / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012492:Salts / Q000494:pharmacology*; D012965:Sodium Chloride / Q000494:pharmacology; D012996:Solutions / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents; D014158:Transcription, Genetic / Q000187:drug effects*
D000085:Acetates; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D009707:Nucleosomes; D012492:Salts; D012996:Solutions; D013439:Sulfhydryl Reagents; D012965:Sodium Chloride; C006421:1,5-I-AEDANS; D012319:RNA Polymerase II; D019342:Acetic Acid