The overall goal of this procedure is to isolate SEC TED 70 s ribosome bound, nascent chain complexes or RNCs produced during translation in an in vitro cell-free system. This is accomplished by first introducing a 17 amino acid long SEC M stalling sequence to the C terminal end of the gene of interest and cloning it downstream of the T seven transcriptional promoter. Next, the mRNA is transcribed using an in vitro transcription reaction and then purified.
Then the purified mRNA is translated in an S 30 E coli extract system with radioactive amino acids to label the synthesized protein or nascent polypeptide. Finally, the second Mr.Ed ribosome bound nascent chain complexes are isolated by sucrose, gradient, centrifugation, and fractionation. Ultimately, results can be obtained showing that nascent chains are stably bound to the 70 s ribosome by gel electrophoresis analysis of the labeled polypeptides isolated from the gradient fractions.
The main advantage of this technique over existing methods like the use of mRNA lacking stock codon is that in it ensures with high efficiency the attachment of nascent polypeptide chain to 70 s ribosome during translation and subsequent centrifugation step. Additionally, in contrast to no stop cordon technology, this method can be used with almost equal efficiency during both in vitro and in vivo system. This method can help answer key questions in the field of tranlational protein folding, such as how early during the translation various tranlational folding intermediates might form and further help to analyze their structure using direct or indirect structure.
Probing and determination approaches This technique can help us to decipher the pathway of protein folding on the ribosome. In this protocol, we have described the isolation of 70 s ribosome bound full length bine gamma B crystalline nascent chains. But this technique can be used for the isolation of vari disease of virtually any protein of interest.
This strategic can also be adopted to isolate truncated natin polypeptide chains of the derm Es.Generally individual new to this method may struggle during the sucrose gradient centrifugation step. However, for experience one, this should not be a problem. We decided to utilize second stalling sequence to isolate gamma B crystalline nascent polypeptides bound to 70 as ribosome after initial, less successful attempts that involved use of mRNA lacking stop cordon.
The later technique could not ensure the stability of RNCs during subsequent centrifugation steps with the same efficiency as the technique involving the use of SECM arrest sequence. Visual demonstration of this method is critical as the steps involving preparation of gradient and separation of RNC may be difficult to learn and it requires certain level of expertise to ensure reproducible results Demonstrating the posture will be graduate student from my laboratory To carry out in vitro transcription and translation. Begin with a gene of interest that is cloned into any T seven and or SP six based plasmid to generate ribosome nascent chain complexes or RNCs.
Extend the C terminus of the target polypeptide by adding the arrest inducing sequence from M in order to ensure that the polypeptide fragment will extrude out of the ribosomal tunnel. Add a flexible glycine Syrian rich linker between the protein and SEC M arrest sequence to prepare for in vitro transcription, user restriction enzyme that will cut downstream of the open reading frame to linearize the template DNA run AROS gel electrophoresis to verify complete digestion of the sample. After verifying the optimum DNA concentration, use a high yield transcription kit according to the manufacturer's instructions to synthesize messenger RNA.
Use lithium chloride precipitation to purify the mRNA. Then run a sample on an acrylamide or aros gel to verify its purity. For in vitro translation.
Using an e coli system in nuclease free water, add a one millimolar amino acid mixture minus methionine 10 units of ribonuclease inhibitor 20, micro curies of 35 s methionine, 20 microliters of reaction mix 24 microliters of reconstitution buffer and 24 microliters of e coli.Lysate. Prewarm the reaction by incubating it at 30 degrees Celsius for five minutes. Then add one to two micrograms of mRNA and incubate for an additional 10 to 15 minutes at 30 degrees Celsius.
Stop the reaction by placing it on ice to isolate 70 s ribosome bound nascent chain complexes or ncs. Layer the translation reaction on top of 4.5 milliliters of a five to 30%sucrose gradient in 20 millimolar heaps. Potassium hydroxide pH 7.5 15 millimolar magnesium, 100 millimolar potassium acetate, and one millimolar DTT centrifuge at 41, 000 RPM for two hours at four degrees Celsius following centrifugation, fractionate the sucrose gradient using a programmable density gradient system and an absorbance detector set at 254 nanometers.
Collect the fractions containing 70 s ribosomes for further analysis to determine whether the SEC M extended protein remains attached to the 70 s ribosome. Precipitate the protein in each gradient fraction by adding trichloroacetic acid. Incubate the samples overnight at four degrees Celsius the next day, centrifuge the samples at 14, 000 times G for 15 minutes to pellet the protein, wash the pellets in a four to one ratio of acetone to one millimolar tris, HCL pH 7.6.
Air dry the pellets, then resuspend them in SDS page loading buffer. Resolve the samples by tris Trice SDS page, then fix and dry the gels and subject them to auto radiography and phospho imaging as shown here. Following in vitro translation, the 70 S ribosomes were isolated by sucrose, gradient, centrifugation, and fractionation to ensure that the gamma B crystalline protein remains stably bound to the ribosome.
The 70 s containing fractions were pooled desalted buffer exchanged and subjected to an additional round of sucrose gradient centrifugation. The results presented here suggests that SEC M can efficiently induce translational arrest of the gamma B crystalline RNCs. Once master, this technique can be done in one working day with exception of gel electrophoresis analysis of nascent chain, which usually involves an overnight TCA precipitation step.
While attempting this procedure, it is obviously critical to avoid RNAs contamination at any steps. RNS contamination may affect translational efficiency of extract and lead to release of nascent polypeptide from the 70th ribosome. Following this procedure, one can obtain NAST and polypeptides of predetermined lanxess stably bound to the 70th ribosome depending on the final goal.
This complexes can be used for various purposes like determination of the confirmation of the ribosome bound nascent chains using direct and indirect approaches like NMR or fre, or testing factor and ligand binding After its development. This technique paved the way for researchers in the field of structural biology and protein folding to explore the confirmation of full length proteins bound to ribosome, as well as cot translational folding intermediates. After watching this video, you should have a good understanding of how to prepare and isolate nascent polypeptide, stably bound to 70 s ribosome, which can be further used to answer various question in field of tranlational protein folding.
Don't forget that working with radioactive isotopes and labeled amine acids, such as, for example, A 35 methionine requires special precautions and should always be taking into account while performing this procedure.
