Measuring the Kinetics of mRNA Transcription in Single Living Cells

Summary

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

Abstract

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.^1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.⁴ Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.^{5, 6} Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.^{7, 8} The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.⁹ To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.⁵ In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

Protocol

The cell system used must contain an integrated gene construct that includes MS2 repeat sequences for tagging the mRNA in living cells. In this protocol we use a human U2OS cell line harboring a stably integrated β-actin gene, that is fluorescently labeled at the DNA, RNA and protein levels⁸, as follows (Figure 1A): The 5’ of the gene contains lac operator (lacO) repeats – these are used to tag the gene (DNA) and thereby identify the genomic locus of integration. A fluorescently tagged lac repressor protein (LacI) will bind to the lacO repeats and tag the DNA. The mRNAs are tagged with YFP-MS2 proteins that bind to the MS2 repeats....

Discussion

The method for measuring polymerase kinetic activity in living cells can be divided into two parts. The first part describes the 'wet' procedure which enables the measuring and retrieving of the kinetic data of mRNA transcription, whereas in the second part, the data is analyzed using an ODE model.⁵ A number of studies have now utilized this approach for extracting transcription kinetics in living cells^{5, 6, 8, 12-14}. The major difference between these studies was the way the FRAP recovery curve was.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Doxycycline	Sigma-Aldrich
FuGENE 6 transfection reagent	Roche Group