The overall goal of this procedure is to identify and locate major translational pause sites for an mRNA translated in a cell-free translation system. This is accomplished by first cloning the gene of interest under the control of a T seven transcriptional promoter. Next mRNA is transcribed and purified.
Then in vitro translation is performed in the presence of mRNA and radioactive amino acids to label the synthesized protein and nascent polypeptides. Finally, the polysome are isolated using sedimentation velocity centrifugation, and the isolated nascent chains are resolved by gel electrophoresis. Ultimately, results can be obtained that show nascent polypeptides of different lengths by Tristine SDS page and auto radiography, thus indicating the presence of the pause sites along the mRNA of interest.
The main advantage of this technique of our existing methods like micro cocal nucleus protection assay is that extremely simple, fast and efficient, and allows determination and identification of the major translation post sites along any MNA in a given cell risk system. This method can help answer some of the key questions in the field of translation and tranlational protein folding such as health factors like cone usage, TNA abundance and or mRNA secondary structure may affect kinetics of translation. This method may further help our understanding of the relationships between the kinetics of translation and the key steps in the code translation protein folding pathway.
The implication of this technique extends towards understanding the process of protein folding in vivo, which is largely believed to occur co translationally during translation on ribosome, though present study involves cell-free translation of gamma bi crystalline using commercially available cell extract. This technique can also be applied to identify translational IDE in potentially any protein using translational competent extract prepared from any cellular source. Generally, individual new to this method will struggle at two key steps of the protocol.
The first one is establishment of the overall time required to run the translation reaction, which would ensure that majority of nascent polypeptides stay bound to the ribosome. And the second one is finding an appropriate just system to resolve Majority of nascent polypeptide, the choice of just system depends on the size of the protein understudy. We first had the idea of this method when we decided to look at the translational kinetics of the globin molecules back in the mid eighties in relation to the proposed model of the COT translation folding of globin.
The non-uniformity of globin translation was first noticed by PRO and Maurice in 1974 when using size occlusion CLO chromatography of the globin NAS and chains. However, their analysis didn't possess the desirable precision Pro and more is observed four major translation power sites during the translation of the globin. Subsequently, our analysis allowed to reveal about 10 additional power sites and build a detailed model of the tranlational globin folding Visual demonstration of this method is critical, as the technique described here involves isolation of polysome and analysis of nascent polypeptide using Tri S Ds page gel.
Both these steps are critical to obtain high quality and reproducible results. Demonstrating the progel will be a graduate student from Myelo laboratory Sujata For in vitro transcription of messenger RNA. Begin with a gene of interest that has been cloned under a T seven and or an SP six promoter using an appropriate restriction enzyme linearize, the DNA template downstream of the stop codon and or MR NA three prime end subject.
A sample of the restriction digestion to aros gel electrophoresis to verify that the DNA is completely linearized. After determining the optimum DNA template concentration, use a transcription kit to in vitro transcribe messenger RNA according to the manufacturer's instructions. Next, use lithium chloride precipitation to purify the mRNA.
Then use either acrylamide or aros gel electrophoresis to verify its purity. Prepare a 100 microliter in vitro translation reaction in nuclease free water by adding a one millimolar amino acid mixture minus methionine 10 units of RNA inhibitor, 20 micro curies of radioactive 35 s methionine and rabbit reticulocyte lysate. Next prewarm the reaction by incubating it at 30 degrees Celsius for five minutes.
Then add two micrograms of mRNA and incubate at 30 degrees Celsius for five minutes to stop the reaction, place it on ice to isolate nascent polypeptides bound to ribosomes. Layer the translation reaction on top of 4.5 milliliters of 30%glycerol interest HCL pH 7.6 with potassium chloride and magnesium chloride using a TLA 110 rotor centrifuge at 100, 000 times G for one hour at degrees Celsius. Next, carefully remove the supernatant, then resuspend the poly somal pellet containing nascent polypeptides in a small volume of one millimolar tris, HCL buffer pH 7.6 containing 0.5 milligrams per milliliter of ribonuclease A and incubate for 30 minutes at 37 degrees Celsius.
To enhance the hydrolysis of the peptide, TRNA Esther bond, add sodium hydroxide at a final concentration of 10 millimolar to the reaction and incubate it for an additional 30 minutes. Resolve the nascent polypeptides on tris tri SDS page, then fix and dry the gels. Subject the gels to auto radiography, followed by phospho imaging for detection of nascent polypeptides.
Altered ribosome movement induced by changes in the TRNA pool should lead to changes in the distribution of sizes of nascent polypeptides accumulating during translation, as well as their relative intensities, which reflect the duration of the pause here. Nascent polypeptides for the gamma B crystalline protein were isolated and resolved by Tristine SDS page. The non-identical lengths and intensities of the polypeptides in the RRL and e coli systems indicates that the movement of ribosomes along mRNA in these two systems follows different translation kinetics.
For example, note the prominent 17.7 kilodalton band in the e coli lysate not present in the rabbit reticulocyte lysate system. Gamma B crystalline harbors, tandem arginine codons that are frequent in mammalian systems, but are known to be extremely rare and slowly translated in e coli, suggesting that this band is caused by ribosome. Pausing at these tandem rare codons as shown here.
No bands can be observed in the absence of mRNA and pur mycin treatment removes most of the nascent chains from polysome. In addition, prolonged treatment with pur mycin removes all the nascent chains confirming that the observed polypeptide products are ribosome associated nascent chains rather than MRPs co sedimenting with polysome Once mastered, this technique can be done in one to two days. The duration of procedure mainly depends on the duration of centrifugation step and nascent peptide gel electrophoresis step.
While attempting this procedure, it is important to stop translation reaction immediately after incubation time. To avoid release of nascent polypeptide from the ribosome, it is obviously critical to avoid RNAs at any step. RNAs contamination may affect the translation efficiency of the extract and lead to release of nascent polypeptide from the ribosome Following this procedure.
Other goals, like for example, the effects of mRNA binding proteins on the efficiency of ribosome movement on a given message can be also tested after its development. This technique paved the way for researchers in the field of protein folding to explore the role of translation posing due to the presence of rare codes in tranlational protein folding. This technique was previously employed to test the role of kaon substitutions on ribosome movement on a message and tranlational protein folding.
After watching this video, you should have a good understanding of how to identify translational power sites presented mRNA by isolating nascent polypeptide bound to ribosome. Don't forget that working with radioactive isotopes and labeled am minor acids, such as, for example, A 35 methionine requires special precautions and should always be taken into account while performing this procedure.
