Fluorescence detection of nucleic acids and proteins in multi-component crystals.
A highly sensitive and specific assay for the rapid detection of nucleic acids in crystals of macromolecular complexes is reported. The assay is based on the observation of crystals under a fluorescence microscope after incubation with a fluorescent dye that specifically binds nucleic acids. The assay allows clear distinction between crystals of free RNA polymerase II and crystals of the polymerase bound to DNA and RNA or RNA alone, even when the nucleic acids account for only 3% of the crystalline material. Fluorescence detection was also used to monitor the diffusion and accumulation of a fluorescence-labelled polymerase-binding protein in preformed polymerase crystals. Additional experiments suggest the general applicability of these fluorescence assays for the characterization of crystal contents.
Kettenberger
Hubert
H
Gene Center, University of Munich, Department of Chemistry and Biochemistry, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
Cramer
Patrick
P
D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D035602:Transcriptional Elongation Factors / Q000378:metabolism
D005456:Fluorescent Dyes; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D012313:RNA; D004247:DNA; D012319:RNA Polymerase II