A fluorescence-based assay for transcription using a novel fluorescent GTP analogue.
A new fluorescent analogue of GTP (Cm-GTP) was synthesized, which contained a coumarin fluorophore attached to the gamma phosphorus. This compound was tested in transcription assays using T7 RNA polymerase as a model system. The fluorescent nucleotide was incorporated specifically at the 5' end of nascent RNA synthesized in two different modes of transcription initiation. In the first mode, with only Cm-GTP (+GTP), reiterative slippage synthesis occurred and poly rG ladders of up to 14 nucleotides were synthesized. In the second mode, with Cm-GTP (+GTP)+ATP+CTP, abortive transcripts of up to seven or eight nucleotides were synthesized. The fluorescence properties of the two types of RNA were studied in detail. There was greater reduction in fluorescence intensity in G-ladders than in abortive transcripts. Steady-state anisotropy and anisotropy decay indicated that the fluorophore motion was constrained in G-ladder RNAs as compared to abortive RNAs. Quenching experiments by using extraneous quenchers showed that the excited state of fluorophore at the 5' end of G-ladder RNA was less efficiently quenched as compared to the free fluorophore. These studies suggested that the fluorescent GTP analogue sensed the structural features that distinguished G-ladder RNAs from abortive RNAs. The results suggested that G-ladder RNAs adapt unusual conformations such as G-quartets. Thus, the new fluorescent probe can be useful for structural studies on RNA.
Sastry
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Laboratory of Molecular Genetics, Box 174, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. sastrys2000@yahoo.com
D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D012313:RNA; D006160:Guanosine Triphosphate