Abstract
The central dogma of biology is performed within this study. A vector of T7 RNA Polymerase gene was inserted into E.Coli where it could be transcribed into RNA and subsequently translated into protein. The protein was extracted and purified. A successful clone was produced by inserting the vector into the expressing cell line and it was observed after achieved an optical density at 600 nm (OD600) of 0.84. A collected 20.87 ug of double stranded RNA was found. Additionally, a linear DNA template OD260: 0.04 µg/ml was transcribed. After expressing it was found that a total protein concentration of protein concentration of 2.03. The T7 RNA polymerase was extracted from this to show a band at 99 kDa. Protein was purified using a several precipitation, an analytical column to obtain a pure fraction. T7 RNA Polymerase is a useful model for showing the central dogma of biology DNA--->RNA ---> Protein.
Introduction
The T7 RNA polymerase is a model protein used for molecular biology applications. Without it molecular biology and applications would not have proceeded at such a fast pace (Steitz, 2004). T7 polymerase is used transcribe DNA that is cloned in vectors and has a very low error rate (Lin, 1999). The molecular weight of T7 polymerase is 99 kDa (Steitz, 2004). The T7 RNA polymerase can transcribe many different types of DNA linked to a T7 promoter, and the T7 expression system and can likely transcribe almost any gene or its complement in E. coli (Studier, 1996). There are many crystal structures available of T7 polymerase which explains how the T7 RNA polymerase initiation and elongation process (Pelletier, 1996; Cheetham, 1999). The enzyme is stimulated by Bovine Serum Albumin or Spermidine (Steitz, 2004).
Figure 1. Illustration of T7 RNA Polymerase showing the DNA (orange) and the RNA being translated (Goodsell, 2003)
In order to obtain purified T7 RNA polymerase three discrete steps need to take place: 1) Transcription 2) Translation 3) Protein Purification. Transcription is where DNA is copied into by RNA polymerase into RNA. In order for T7 RNA polymerase to work the DNA used as a template needs to be linearalized. In most biological organisms this is the first step of gene expression. A complementary antiparallel RNA strand is produced which is called a primary transcript. This strand can be visualized in an agarose gel.
Once the primary strand is transcribed the cell can translate the protein in ribosomes to create proteins. Specifically, the ribosomes decode the messenger RNA (mRNA) and tRNA help this process by carrying specific amino acids to the ribosome. During this process the proteins is moving out from the ribosome and folding into a more native conformation. Once the protein is translated the the ribosomes break off and the protein is transported.
If sufficient quantities of protein is expressed it can be extracted from the E.Coli, purified and characterized. There are several ways to purify proteins depending on their physicochemical properties including: precipitation, filtration, ion exchange, size-exclusion, and affinity chromatography (Katoch, Rajan, 2011). Once the protein is purified it can be measured using spectroscopy and then characterized by a plethora of techniques.
Within a cell, in vitro, RNA transcripts can be generated. One interesting RNA transcript that could be generated using T7 RNA polymerase is that of pokeweed plant (Phytolacca americana) which is a naturally occurring single-chain ribosome-inactivating antiviral protein (Mansouri, 2009). This protein can be used for therapeutic use in different diseases particularly Human T-cell leukemia virus. There is currently, no known cure for this. Interestingly this antiviral protein is a ribosome-inactivating protein that removes a specific adenine base from the large ribosomal RNA a process called depurination. This overall suppresses the synthesis of viral proteins by decreasing the translational efficiency of HTLV. Suppression is done both at the transcriptional and translational levels. Additionally this protein is not so toxic making it a good drug candidate (Mansouri, 2009). Within this study pokeweed plant antiviral protein was transcribed by T7 RNA polymerase to make sufficient quantities.
The DNA of pokeweed plant was transcribed using radionucleotides using a technique called PhosphorImager. Within this technique high sensitivity can be obtained. In brief a luminescent signal is produced from a release of stored energy within a phosphor after stimulation with visible light. It reveals a sensitive image on an image plate or phosphorimager. It is a application in molecular biology to detect radiolabeled RNA (Ekstrand, 2004).
Protein expression is a central part of biology. This experiment will go over the specific details of transcription, translation and protein purification to fundamental understanding the biological dogma using T7 RNA Polymerase as a model protein. Hypothesis: Using linearalized DNA T7 can transcribe pokeweed RNA transcripts.
Materials and Methods
All the materials were provided from the laboratory. The methods were performed according to the details of the laboratory manual.
Results
Protein Purification
Here protein T7 RNA polymerase was purified. A band at 100 kDa can be observed (Fig 1 A, Lane 2-4). Using filtration and successive washes a prominent protein band was observed (Fig B, Lane 5). These faint bands disappear after each wash.
Figure 1 (Upper Panel) Lane 1: Empty; Lane 2 T7 undiluted 15 ul; Lane 3: diluted 1:2 15 ul; Lane 4 T7 diluted 1:10; Lane 5 Protein Marker 5 ul; Lane 7 BSA 0.5 ug (15 ul); Lane 8 BSA 1 ug, 15 ul
Figure 1 B (Lower Panel). Purification of T7. In each lane 15 ul of sample was loaded. Lane 1 : Blank; Lane 2: 1st purification; Lane 3: 2nd purification; Lane 4: 3rd purification; Lane 5: 4th purification; Lane 6: 5th purification; Lane 7: sixth purification; Lane 8: Protein marker; Lane 9: Unknown
The T7 polymerase concentration (Fig 1) was estimated by comparing with known dilutions of BSA on SDS-PAGE stained with Coomassie. The staining intensity of the most purified T7 polymerase was compared with dilution series of BSA staining intensity. The concentration of T7 polymerase is thus equal to 1 µg/µl of BSA of the same intensity.
Transcription
The T7 RNA polymerase enzyme was used to synthesize a single RNA transcript. The T7RNA polymerase was expressed in BL21 bacterial cells. After growing in YT medium the OD600 was found to be 0.84 which corresponded to 20.87 ug/ml of double stranded RNA. The KH014 pBluescript plasmid contained an active site mutant for the pokeweed antiviral protein that is driven by the T7 promoter.
Isolation of Plasmid DNA and linearalized. The plasmid DNA was isolated at an OD260 of 0.04 ug/ml. Using the Beer Lambert Law the concentration of DNA can be measured using spectroscopy and the extinction coefficient depends on the properties e.g. single stranded, double stranded, length and base composition.
Linearalizing DNA
In order to use the plasmid DNA as a substrate for in vitro transcription this had to be linearalized with restriction endonucleases particularly HindIII. Using this technique a linear piece of DNA was found to have an optical density corresponding to 0.04 ug/ml. This was compared to the coiled DNA and supercoiled DNA. Both the coiled and supercoiled DNA travelled through the agarose gel faster because of its more compact size. Linear DNA was approximately 3.7 Kb observed from comparing the ladder. The purpose of doing it was to make sure that we have linear DNA before the transcription. The linear DNA gave a more intense band then the non-linear DNA.
Figure 2 A (upper panel) OD260 of 0.28 ug/ml. In vitro Transcription Lane 1 is Molecular Weight Ladder Lane 2: Positive Control Lane 3: Negative Control Group 3; Lane 4 Experiment Group 3; Lane 5 Negative Control Group 2; Experiment Group 2; Negative Control Group 7; Negative Control Group 7.
Figure 2B (lower panel) the DNA was an OD260 of 0.04 ug/ml Lane 1: DNA Marker; Lane 2: Uncut DNA; Lane 3: Cut DNA.
In vitro transcription
Here the transcript used in section B was used to translate in vitro a radiolabelled RNA that is visualized by phosphoimager.
Bacteriophage T7 RNA polymerase synthesized a single strand RNA which was highly specific for the T7 promoter. The enzyme catalysed reaction is represented by the following expression, the synthesis of RNA being downstream from the promoter and in a 5'--->3' direction.
The scanned images were with a filter for blue (500 nm) and green (520 nm) fluorescence (note: the dye fluoresces using the blue filter for some reason). The ladder is in red and a bright green band at 800 bp is the transcript which was produced.
Figure 3 Lane 1: Molecular Weight Marker; Lane 2: Positive control; Lane 3: Negative Control Group 6; Lane 4: Experiment Group 6; Lane 5: Negative Control Group 1; Lane 6: Experiment Group 1; Lane 7: Negative control Group 5; Lane 8: Experiment group 5
Discussion
Expressing of T7 RNA Polymerase in BL21 bacterial cells is well characterized in several studies (Studier, 1986). Here high concentrations of RNA polymerase and promoter result in RNA transcription from all the protein in the cell. By using low concentrations it was found that T7 polymerase can bind to promoters preferentially. At least 2 ug/ml was achieved to visualize a pronounced band on the gel (Figure 1 A) estimated comparing it with known BSA standard dilutions. BSA standards are an efficient method to measure the total protein in the sample. This procedure allows estimates of protein up to microgram amounts (Ramagli, 1985). This band got stronger with subsequent washes to remove any other proteins from the system (Figure 1 B). There are several different ways to do protein purification. Within this study only a commercial gravity flow column was used to purify the protein and a concentration and the protein was concentrated using Centricon 50 filters which has a molecular weight cutoff. Although this allows efficient desalting and washing of protein at particular molecular weight protein fragments of smaller molecular weight will also be concentrated along with the T7 RNA polymerase. Another size-exclusion chromatography step where fractions are collected at specific times might help to increase the purity but it might be more difficult with the small concentration used here.
A circular piece of DNA was extracted from the bacteria. This result was expected as most bacteria have one or more plasmids that can be extracted. However, within Figure 2A no transcript was observed. Possibly the conditions used to make the transcript using T7 RNA polymerase were not satisfactory. T7 RNA polymerase seems to be capable of transcribing almost any DNA linked to a T7 promoter, so the T7 expression system should be capable of transcribing almost any gene or its complement in E. coli. We expect that comparable T7 expression systems can be developed in other types of cells.
It is efficient at synthesizing small RNAs and has been optimized to synthesize nearly milligram amount of any RNA from 12-35 nucleotides long (Milligan, 1987). A linear piece of DNA is needed for the RNA polymerase with a specific promoter region exposed so RNA polymerase could bind. This was found with a large orange band in figure 2 B. If the DNA is coiled or knotted it will slow down or stop transcription. Coiling or knotting the DNA thus can regulate the rate of transcription (Portugal, 1996). The linear DNA was used to transcribe pokeweed transcript in sufficient quantities (Fig 3).
Using radioactivity pokeweed transcript could be produced using T7 RNA polymerase. Using T7 RNA polymerase promoters on the gene additionally with the T7 RNA polymerase enabled suitable expression within this system. In Figure 3 a 800 bp sequence was synthesized with a large green band. The phosphoimage technique is useful as the gels can be visualized again as it only is bleached when exposed to UV light. This makes this technique quite useful for visualizing RNA or other biomolecules in small quantities.
The technique for amplification is used for in vivo cancer gene amplification and is called RNA PCR as it achieves high fidelity, purity, specificity and reproducibility (Lin, 1999). Using this technique a lot of RNA can be synthesized. T7 RNA polymerase is efficiently synthesize the RNA of pokeweed antiviral protein.
How could these results be used as a therapeutic? As mentioned Pokeweed antiviral proteins are very interesting and can disrupt ribosomes. Pokeweed antiviral protein is a difficult protein to synthesize in vitro as it disrupts both prokaryotic and eukaryotic transcription (Rajamohan, 1999). Two strategies could be proposed that would enable this to be used as a therapeutic However, with sufficient quantities of the RNA produced using T7 RNA polymerase this could be inserted into the body and into cells. By adding it to the host cells it could be transcribed by the host and the protein will silence the host ribosomes. This would effectively be RNA silencing. Secondly, protein could be synthesized externally and inserted into the organism. If it can penetrate the cell it would be able to stop the ribosomes directly. These two strategies could be utilized in treating several diseases including viruses where ribosomes are used to generate viral proteins or any other proteins for that matter.
This experiment allowed efficient expression of T7 RNA polymerase a nucleotidyl transferase that is used in transcription. A purification method was used to isolate this protein to be utilized later. The T7 RNA polymerase could transcribe nearly a linear piece of DNA with a promoter attached. T7 RNA polymerase was used to transcribe RNA in sufficient quantities that encodes a protein called pokeweed antiviral protein. The protocol for this experiment could be used to make sufficient quantities of specific RNA sequences. Fundamentally, it could be used to understand the central dogma of biology better as all processes form transcription to translation were captured here.
Conclusion
Within this paper T7 RNA Polymerase was expressed and purified. DNA was linearalized and utilized to make multiple RNA sequences. This paper shows a method to understand the central dogma in biology.
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