The overall goal of the following experiment is to analyze the structure and function of pure ribosomes containing a unique peptid TRNA. This is achieved by performing in vitro translation assays with release factor depleted, cell-free extracts and biotin labeled mRNAs to generate stalled pep tRNA ribosome complexes attached to the biotin labeled mRNAs. As a second step, streptavidin bound paramagnetic beads are mixed with the in vitro translation assays to allow their interaction with the biotin molecules located in the mRNA.
Later, the beads are extracted using magnetic fields which pulled the stalled peptid tRNA ribosome complexes attached to the mRNAs out of the total reaction. Next, the purified peptid tRNA ribosome complexes can be mixed with translational factors such as release factor translation, inhibiting antibiotics or chemical modifying agents in order to analyze the function. Inhibition and structure of the ribosome Results are obtained that show inhibition of hydrolysis of the peptide tRNAs and its correlation with structural changes in the ribosomes based on peptide transferase activity or translational termination assays using SDS or Tris Racine page gels and modification protection assays using urea page gels.
The main advantage of this technique over existing methods like differential centrifugation, is that the isolated samples mostly contain ribosomes with only one population of pep tRNA. This method can help to answer the key questions in the gene expression field, such as the nature of nascent polypeptides and antibiotics affect the ribosome function like modulating the translation process. To begin this procedure, obtain two grams of a dry e coli bacterial pellet from log phase cultures.
Wash the pellet by Resus suspending in one liter of buffer, a followed by centrifugation at 5, 000 Gs for five minutes at four degrees Celsius. After performing two washes, resus suspend the resulting bacterial pellet in 40 milliliters of buffer a disrupt the bacterial cells using a French press applying 6, 000 PSI pressure. This pressure corresponds to 600 units Using a one inch piston.
Collect the disrupted suspension in a 50 milliliter clean glass tube. Treat the disrupted suspension with one millimolar DIO triol or DTT centrifuge the sample at 30, 000 Gs for 30 minutes. Remove the resulting supernatant and repeat the centrifugation procedure.
Save the supernatant obtained from the second centrifugation, dispense the final supernatant into micro tubes. Finally, flash. Freeze the aliquots using a dry ice ethanol mixture or liquid nitrogen and store the samples at minus 60 or minus 80 degrees Celsius.
Begin the preparation of cell-free extracts depleted of RF two by mixing 4.5 milliliters of protein A spheros four B slurry beads with five milliliters of an anti RF two antiserum. Rotate the mixture on a rotating wheel at room temperature for one hour. Centrifuge the mixture at 2, 500 Gs to separate the beads from the antiserum.
Discard the supernatant. The beads now contain the anti RF two antibodies and will be referred to as anti RF two beads. Wash the beads by Resus suspension in one milliliter of buffer bee.
Then centrifuge the beads as before to separate the anti RF two beads from buffer B.Repeat this washing procedure twice. After washing the beads, mix one milliliter of the cell-free extract with 150 microliters of anti RF two beads and incubate on a rotating wheel at four degrees Celsius for two hours following incubation. Centrifuge the mixture at 10, 000 Gs to separate the cell-free extract from the beads.
Remove the supernatant and treat it again with another 150 microliters of anti RF two beads before repeating the incubation and pull down once more. Dispense the final RF two depleted cell-free extract solution into micro tubes at 100 microliters per tube. Freeze the aliquots using a dry ice ethanol mixture or liquid nitrogen and store at minus 60 or minus 80 degrees Celsius.
Prepare the PCR reaction by combining the plasmid template containing the sequences to be translated oligo deoxy nucleotides DTPs intact DNA polymerase in strategy PCR buffer. Once the PCR sample has been prepared, perform the amplification reaction precipitate the DNA in order to purify the PCR products by adding one 10th volume of three molar sodium acetate pH 5.2 and two volumes of ice cold ethanol. After repeating the precipitation procedure once more, we suspend the resulting DNA in 100 microliters of dathyl percarbonate or dsy treated water.
Verify the integrity of the PCR products by electrophoresis on agro gels. Typically, this procedure yields 100 micrograms of a 600 base pair DNA product. Prepare a 100 microliter in vitro transcription reaction in depthy treated water by mixing five micrograms of the PCR generated DNA fragment with ribonucleotides biotin labeled UTP and T seven enzyme mix.
Incubate the reaction mixture at 37 degrees Celsius for three hours. To quantify the amount of mRNA obtained, eliminate the DNA template by adding RNA free D ns. Incubate the reaction at 37 degrees Celsius for 10 minutes.
To purify the mRNA products, precipitate the RNA twice by adding one 10th volume of three molar sodium acetate pH 5.2 and two volumes of cold ethanol resuspend the precipitated mRNA in 100 microliters of dpci treated water. Verify the integrity of the biotin labeled mRNA products by electrophoresis on aros gels. Usually this procedure yields between one to two milligrams of a 600 nucleotide biotin labeled mRNA product to begin the isolation of translating ribosomes containing a peptid TRNA 500 microliter in vitro translation reaction mixture is prepared in dey treated water.
First, prepare the buffered reaction mixture described in the accompanying written protocol, and then add 75 M of each of the amino acids except the one that will be replaced by a radioactive amino acid. Next, add 20 to 50 microliters of the RF two depleted cell-free extract to the buffer solution containing the amino acids. Incubate the mixture for five minutes at room temperature to allow the activation of the ribosomes.
Once the ribosomes have been activated. Add 10 to 15 micrograms of biotin labeled mRNAs to the sample. Incubate the reaction mixture at 37 degrees Celsius for 10 minutes.
Meanwhile, prepare three milliliters of streptavidin paramagnetic beads or SMB in buffer C.Following incubation, add the SMP suspension to the translation reaction mixture. Incubate the new suspension at room temperature for 10 minutes. Separate the SMB from the mixture by applying a magnetic field using magnetic separation stands.Resus.
Suspend the SMB in buffer C and separate the beads again using the magnetic stand. After repeating this washing procedure twice, resuspend the beads in 500 microliters of buffer C.Store the suspension on ice and perform the next procedure immediately. To visualize the peptid TRNA, mix 10 microliters of the SMB suspended and buffer C with 10 microliters of loading buffer, resolve the components attached to the beads by running the samples in 10%Tris tric poly acrylamide gels.
Dry the gels using a vacuum gel dryer. Then verify the integrity and purification of the peptide TRNA by exposing the dried gel to an x-ray film. To analyze the ribosomal RNA, combine 190 microliters of two millimolar EDTA solution prepared with dpci treated water, 200 microliters of phenol equated with water and 10 microliters of a bead suspension.
Mix the resulting suspension by vigorous vortexing. Separate the inorganic phase from the organic phase by centrifugation at 10, 000 Gs for three minutes At room temperature, collect the top water layer in a new micro tube, precipitate the RNA from the water layer by adding one 10th volume of three molar sodium acetate pH 5.21 microliter of 20 milligrams per milliliter glycogen solution, and two volumes of ice cold ethanol. Re suspend the precipitated RNA in 10 microliters of dpci treated water.
Verify the integrity of the ribosomal RNAs by electrophoresis on niros gels. Usually 50 microliters of beat suspension yields one microgram of ribosomal RNA. Here the results of a series of analysis evaluating the quality and functionality of isolated translating ribosomes are presented.
The observation of a unique band resolved in poly acrylamide gels indicates the presence of polypeptides bound to the biotin labeled mRNAs attached to the SMB. The purification of ribosomal RNAs. Using this procedure establishes the presence of ribosomes bound to these biotin labeled mRNAs as well.
Addition of pur mycin an antibiotic that induces the peptid transferase activity of the ribosome results in cleavage of the nascent peptid tRNAs. This is observed as a shift in the migration pattern of the isolated polypeptide in poly acrylamide gels. Together these data indicate that biotin labeled mRNAs attached to the SMB contain functional ribosomes with peptid tRNAs.
The isolation of translating ribosomes containing specific peptid tRNAs permits the study of the effects of peptid tRNAs antibiotics and other molecules on ribosome function and on ribosome structure. In the examples presented here, the antibiotic s parsamyn and the amino acid trytophan inhibit the hydrolytic cleavage of the nascent TNAC tRNA peptid tRNA by RF two. In the isolated ribosomes.
The interaction of trytophan or spars mycin with the ribosome also induces structural changes in some nucleotides that constitute the 23 S-R-R-N-A of the translating ribosome. Once mastered, the main part of this technique can be done in five hours of performed properly Following this procedures. Other methods like cryo em are extra structural analysis can be performed to answer additional questions about the structure of ribosome containing unique Pepin, RNA.
