A3 Refereed book chapter or chapter in a compilation book
Fluorogenic RNA aptamers to probe transcription initiation and co-transcriptional RNA folding by multi-subunit RNA polymerases
Authors: Huang Yong-Heng, Trapp Vilma, Puro Oskari, Mäkinen Janne J., Metsä-Ketelä Mikko, Wahl Markus C., Belogurov Georgiy A.
Publisher: Academic Press Inc.
Publication year: 2022
Book title : Methods in Enzymology
Journal name in source: Methods in Enzymology
Series title: Methods in Enzymology
Volume: 675
First page : 207
Last page: 233
ISSN: 0076-6879
DOI: https://doi.org/10.1016/bs.mie.2022.07.010(external)
Transcription is the first and most highly regulated step in gene expression. Experimental techniques for monitoring transcription are, thus, important for studying gene expression and gene regulation as well as for translational research and drug development. Fluorescence methods are often superior to other techniques for real-time monitoring of biochemical processes. Green fluorescent proteins have long served as valuable tools for studying the process of translation. Here we present two methods that utilize fluorescent light-up RNA aptamers (FLAPs), the RNA mimics of green fluorescent proteins, to monitoring transcription and co-transcriptional RNA folding. FLAPs adopt defined three-dimensional folds that bind low molecular weight compounds called fluorogens with concomitant increase in fluorescence by many folds. FLAPs provide a strong fluorescence signal with low background that allows monitoring of transcription in real time in vitro and in vivo. However, it takes several seconds for RNA polymerase to synthesize FLAPs and the subsequent folding of the fluorogen-binding platform takes additional seconds or minutes. Here we show that Broccoli-FLAP is well suited for monitoring the rate of transcription initiation in a multi-round setup that mitigates the slow rate of the FLAP maturation. Furthermore, we demonstrate that a relatively slow and inefficient folding of iSpinach-FLAP can be taken advantage of for monitoring the action of RNA folding chaperones.