A fluorescence-based assay for transcription using a novel fluorescent GTPanalogue

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 synthes is occurred and poly rG ladders of up to 14 nucleotides were synthesized. I n the second mode, with Cm-GTP (+ GTP) + ATP + CTP, abortive transcripts of up to seven or eight nucleotides were synthesized. The fluorescence proper ties of the two types of RNA were studied in detail. There was greater redu ction 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. Quen ching experiments by using extraneous quenchers showed that the excited sta te of fluorophore at the 5 ' end of G-ladder RNA was less efficiently quenc hed as compared to the free fluorophore. These studies suggested that the f luorescent GTP analogue sensed the structural features that distinguished G -ladder RNAs from abortive RNAs. The results suggested that G-ladder RNAs a dapt unusual conformations such as G-quartets. Thus, the new fluorescent pr obe can be useful for structural studies on RNA. (C) 2001 Elsevier Science B.V. All rights reserved.