The overall goal of the following experiment is to measure RNA polymerase two transcriptional dynamics in living cells as it transcribes from a specific gene in real time. In order to follow transcription on a specific gene sequence, a gene construct is stably integrated into a human cell line. This gene enters in several copies into one random site in the genome and forms a gene locust containing a tandem array of this specific gene.
When gene expression is activated, RNA polymerase two is recruited to the gene and moves downstream along the DNA strand, generating the mRNA molecule in order to tag and detect the transcribed mRNA. A series of unique sequences or MS two repeats that serve as binding sites for a fluorescent protein YFP MS two have been fused into the gene sequence. The rate at which the MS two STEM loops are formed corresponds to the rate of transcription fluorescence recovery after photobleaching or FP is then used to visually detect ongoing transcription.
A strong laser beam is directed at the active transcription site to photobleach the YFP MS two molecules, and the recovery of fluorescence is followed. The rate at which the fluorescence signal recovers reflects the rate at which RNA polymerase two is transcribing. A recovery curve is obtained that shows the measurement of the increase in YFP MS two intensity at the transcription site.
Over time, the transcriptional kinetics can then be extracted from these data. The results obtained show that RNA polymerase two elongation rates in vivo can be measured and that these are much faster than previous in vitro measurements. The main advantage of this technique over existing methods like biochemical approaches, is that here we can measure kinetics within a single living cell on a specific gene.
These methods can provide insight into the most basic process of mRNA transcription occurring in each and every cell of our body. Visual demonstration of these methods is important as there are several steps that first involve. We experiment with cells under a microscope, and these need to be followed by data analysis using computer software In order to analyze RNA polymerase two elongation kinetics on a specific gene in living cells.
This protocol uses a human U2 OS cell line, in which a stably integrated beta actin gene it's mRNA product, and the final protein product can be fluorescently labeled. Shown here is a schematic representation of the CFP beta actin gene construct that was integrated into the U2 OS cells. Transcriptional induction from the minimal CMV promoter is achieved by the binding of reverse tetracycline transcriptional activator to tet responsive elements or tres in the presence of doxycycline, the transcribed mRNA contains the coating sequence for CFP Beta Acton and 24 MS two repeats that are bound by the YFP MS two fusion protein.
An active transcription site will appear yellow due to the binding of the YFP MS two proteins. The acton cytoskeleton will appear blue due to the CFP tagged Acton 48 hours before the fluorescence recovery after photobleaching or F frappe experiment, prepare the U2 OS cells for transfection seed approximately two times 10 to the fifth cells on glass bottomed tissue culture dishes, and add two milliliters of medium to the cells in order to reach 50 to 80%Confluence incubate the cells overnight at 37 degrees Celsius 24 hours later, 24 hours before the frack experiment, transiently transfect the cells with a YFP MS two construct that expresses the fluorescent marker for tagging the mRNA. The YFP MS two construct contains a nuclear localization sequence or NLS that provides nuclear entry for the YFP MS two protein.
Incubate the cells for at least 12 hours at 37 degrees Celsius to obtain good expression of the transient transfected proteins. Six to 12 hours before the experiment, add doxycycline to the cells for activating the TED inducible CFP actin gene return cells to the 37 degrees Celsius incubator. Since our experiment is relatively long and we are following one small locus within the nucleus, it is important to be able to keep the gene locus transcription site in focus throughout the whole experiment.
This required rapid imaging in three dimension over time. Therefore, we use a 3D FRA system instead of a confocal laser scanning microscope. Half an hour before the live cell experiment turn on the heating device microscope and laser.
The equipment used for this demonstration are a 3D frap system built on a wide field, Olympus IX 81 microscope, equipped with an EM CCD that can acquire images at high speed up to 30 images per second. The microscope is equipped with a Lambda DG four light source and an x, y, and Z stage that allows fast and accurate imaging. In the Z axis, all equipment is integrated and controlled by metamorph software.
The experiment is performed at 37 degrees Celsius with 5%CO2 using a live cell chamber system. To avoid focus changes during the long acquisition times, the temperature must remain constant During the experiment. After the laser and camera have been synchronized, a good candidate cell must be chosen for the experiment.
A good cell should a exhibit A clear YFP MS two tag transcription site above the diffuse YFP MS two background, and B, have enough free YFP MS two protein in the cell so as to be able to recover completely after the photo bleaching. Next, choose appropriate measuring conditions. The frat procedure requires the acquiring of many images.
Therefore, it is important to balance between reasonable exposure times and using as low light as possible. To minimize photobleaching cells in the YFP channel should be imaged with 150 millisecond exposure times for each acquisition. Seven Z slices will be taken every 350 nanometers.
The active transcription site will be bleached using the 491 nanometer laser. Post bleach images will be acquired in a sequence of three time frequencies, 15 images every three seconds, 15 images every six seconds, and 26 images every 30 seconds. After a suitable cell and appropriate measurement conditions have been selected, perform the full experiment but without bleaching to verify that the act of transcription site is functional and stable at steady state.
This tests the imaging conditions by measuring the total photobleaching caused by the full imaging series. Next, perform the frab experiment. First, acquire six preble images.
Then use the 491 nanometer laser to bleach the YFP MS two signal at the active transcription site and acquire post bleach images in a sequence of three time frequencies. 15 images every three seconds, 15 images every six seconds, and 26 images every 30 seconds. The four D frap data is analyzed using the free image analysis software.Image.
JZ stacks are maximum projected at every time point resulting in a 2D movie over time. For further analysis, the transcription site is tracked in each frame and the mean intensity is measured. Using the spot tracker tool, the data is saved in an Excel sheet for further analysis using the time series analyzer, plugin of image J.Measure the mean intensities at other regions of interest or ROIs for every time point as follows, a measure the background from an ROI outside of the cell B measure an ROI in the nucleoplasm that as far as possible from the bleach site and C, measure the transcription site after correcting for photobleaching during imaging, normalize the data for at least 10 experiments in order to retrieve the kinetic parameters from this kind of dataset.
The simplest described model that can fit this kind of data is used shown here is the scheme of the model describing the entrance and exit kinetics of mRNAs marked with YFP MS two molecules at the transcription site. This model contains the following equations that describe the scheme. Having the model parameters, it is possible to calculate the elongation rate using this equation.
Shown here is an example of a cell in which an active transcription site was photo bleached, and the fluorescence recovery was tracked over time. The fluorescence intensity of both bleached and non bleached active transcription sites was measured. This graph shows the experimental measurements performed on the CFP ACT and gene transcription sites.
The recovery curves of the YFP MS two FP measurements are in blue measurement of YFP MS two signal at the transcription site. Over time in a non bleached cell from the same experiment, gave a constant signal indicated by the red dots, which implies that this gene is currently in a relative steady state. The recovery curve of the YFP MS two frap measurement was analyzed using Berkeley Madonna to generate this fitted simulation.
The calculated residuals of the fit are presented at the bottom. Finally, the model results are shown in this parameter table. This method can help answer key questions in the transcription field, such as what or the in vivo rates of transcription and which cot transcriptional processes affect the kinetics.
After watching this video, you should have a good understanding of how to follow the transcriptional process in real time. This technique can be expanded to follow the kinetics of other transcription factors or MNA processing factors on actively on actively transcribing genes.
