In biology, a codon refers to the trinucleotides that specify for a particular amino acid. What is the Genetic Code? Come join us now! The copy of a DNA segment for gene expression is located in its coding region.
It consists of two major sites: 1 anticodon arm and 2 acceptor stem. The anticodon arm contains the anticodon that complementary base pairs with the codon of the mRNA. The acceptor stem is the site where a specific amino acid is attached in this case, the tRNA with amino acid is called aminoacyl-tRNA.
Rather, it serves as one of the components of the ribosome. The ribosome is a cytoplasmic structure in cells of prokaryotes and eukaryotes that are known for serving as a site of protein synthesis. The ribosomes can be used to determine a prokaryote from a eukaryote. Prokaryotes have 70S ribosomes whereas eukaryotes have 80S ribosomes. Both types, though, are each made up of two subunits of differing sizes.
The larger subunit serves as the ribozyme that catalyzes the peptide bond formation between amino acids. The A aminoacyl site is where aminoacyl-tRNA docks. The P peptidyl site is where peptidyl-tRNA binds. The E exit site is where the tRNA leaves the ribosome. Transcription is the process by which mRNA template , encoding the sequence of the protein in the form of a trinucleotide code, is transcribed from DNA to provide a template for translation through the help of the enzyme, RNA polymerase.
Thus, transcription is regarded as the first step of gene expression. But unlike DNA replication, transcription needs no primer to initiate the process and, instead of thymine, uracil pairs with adenine.
The steps of transcription are as follows: 1 Initiation, 2 Promoter escape, 3 Elongation, and 4 Termination. The first step, initiation, is when the RNA polymerase, with the assistance of certain transcription factors, binds to the promoter of DNA. This leads to the opening unwinding of DNA at the promoter region, forming a transcription bubble. A phase of abortive cycles of synthesis occurs resulting in the release of short mRNA transcripts about 2 to 15 nucleotides.
The next step is for the RNA polymerase to escape the promoter so that it can enter into the elongation step. During elongation, RNA polymerase traverses the template strand of the DNA and base pairs with the nucleotides on the template noncoding strand. In eukaryotes , the new mRNA is not yet ready for translation. At this stage, it is called pre-mRNA, and it must go through more processing before it leaves the nucleus as mature mRNA.
The processing may include splicing, editing, and polyadenylation. These processes modify the mRNA in various ways. Such modifications allow a single gene to be used to make more than one protein. It is the process in which the genetic code in mRNA is read to make a protein.
Translation is illustrated in Figure 5. The ribosome reads the sequence of codons in mRNA, and molecules of tRNA bring amino acids to the ribosome in the correct sequence.
After transcription in the nucleus, the mRNA exits through a nuclear pore and enters the cytoplasm. At the region on the mRNA containing the methylated cap and the start codon, the small and large subunits of the ribosome bind to the mRNA. As a tRNA moves into the ribosome, its amino acid is transferred to the growing polypeptide. Once this transfer is complete, the tRNA leaves the ribosome, the ribosome moves one codon length down the mRNA, and a new tRNA enters with its corresponding amino acid.
This process repeats and the polypeptide grows. At the end of the mRNA coding is a stop codon which will end the elongation stage. After a polypeptide chain is synthesized, it may undergo additional processes.
For example, it may assume a folded shape due to interactions between its amino acids. It may also bind with other polypeptides or with different types of molecules, such as lipids or carbohydrates.
Many proteins travel to the Golgi apparatus within the cytoplasm to be modified for the specific job they will do. In fact, every amino acid is represented by a three-nucleotide sequence or codon along the mRNA molecule.
Figure 7: The ribosome and translation A ribosome is composed of two subunits: large and small. During translation, ribosomal subunits assemble together like a sandwich on the strand of mRNA, where they proceed to attract tRNA molecules tethered to amino acids circles. A long chain of amino acids emerges as the ribosome decodes the mRNA sequence into a polypeptide, or a new protein. Each tRNA molecule has two distinct ends, one of which binds to a specific amino acid, and the other which binds to the corresponding mRNA codon.
During translation , these tRNAs carry amino acids to the ribosome and join with their complementary codons. Then, the assembled amino acids are joined together as the ribosome, with its resident rRNAs, moves along the mRNA molecule in a ratchet-like motion.
The resulting protein chains can be hundreds of amino acids in length, and synthesizing these molecules requires a huge amount of chemical energy Figure 8.
Figure 8: The major steps of translation 1 Translation begins when a ribosome gray docks on a start codon red of an mRNA molecule in the cytoplasm. A second tRNA molecule, bound to two, connected amino acids, is attached to the 4 th , 5 th , and 6 th nucleotide from the left.
It no longer has amino acids bound to its terminus. In step 4, the tRNA molecule that formerly had two connected amino acids attached to its terminus, has now accumulated four amino acids total. Different colored spheres represent different amino acid types, and the four spheres are connected end-to-end in a chain.
A tRNA to the right has one amino acid attached to its terminus. A tRNA molecule carrying a single amino acid is shown approaching the ribosome from the cytoplasm. In step 5, the ribosome is shown to have moved along the length of the mRNA molecule from left to right. A long chain of approximately 19 amino acids is connected to the end of the tRNA molecule. Five tRNA molecules carrying a single amino acid each are seen floating freely in the cytoplasm surrounding the mRNA molecule.
In step 6, the ribosome is disassociated from the mRNA molecule. The amino acid chain has disassociated from the tRNA and is floating freely in the cytoplasm as a complete protein molecule.
The illustrated ribosome is translucent and looks like an upside-down glass jug. The mRNA is composed of many nucleotides that resemble pegs aligned side-by-side along the molecule, in parallel. Each type of nucleotide is represented by a different color yellow, blue, orange, or green. The first three nucleotides, bound to the ribosome, are highlighted in red to represent the stop codon. In step 2, a tRNA molecule is bound to the stop codon. At the end of the tRNA molecule opposite this point of attachment is an amino acid, represented as a sphere.
In step 3, a tRNA bound to a single amino acid is attached to the 7 th , 8 th , and 9 th nucleotide from the left. In eukaryotic cells, however, the two processes are separated in both space and time: mRNAs are synthesized in the nucleus, and proteins are later made in the cytoplasm.
This page appears in the following eBook. Aa Aa Aa. Ribosomes, Transcription, and Translation. Figure 1: DNA replication of the leading and lagging strand. The first lot of phages began to be synthesized following 1 h of incubation and their accumulation continued for 5 h.
It was also reported that the addition of dNTPs increased the phage production by nearly fold and further investigations revealed that after the fourth hour of incubation, genomic DNA began to degrade, contributing to the observed arrest in synthesis. Even though a cytosolic lack of thioredoxin tends to impair DNA replication in vivo , it was highlighted that the absence of thioredoxin in these cell-free TX—TL systems does not impede phage production Shin et al.
These bio-nanomaterials are devoid of genetic information and as such cannot self-reproduce, but due to their ability to display high-density viral surface proteins, they may successfully penetrate into a living cell. These particles are formed during the heterologous expression of viral proteins of the same or different viruses in a system, or spontaneously during the viral life cycle inside a cell Chroboczek et al.
These empty shells lacking a viral genome have the potential to be used as safe vaccines because of their ability to elicit an immune response and lack of self-replication. Stimulation of innate immunity by VLPs is facilitated through pattern recognizing receptors and toll-like receptors and the induction of a strong humoral response. This is augmented further through better uptake, processing, and presentation by antigen-presenting cells, due to highly specific structures and multimeric antigens of VLPs Shirbaghaee and Bolhassani, Aside from vaccination, VLPs have gained interest in fields of gene therapy, drug delivery, nanotechnology, and diagnostics Shirbaghaee and Bolhassani, In conventional cell-based VLPs production, they were produced in vivo and their assembly was separated from the large pool of proteins ex vivo.
Numerous difficulties are faced in conventional cell-based system such as poor yields, low solubility of the bacteriophage proteins, lack of post-transcriptional modifications, complications in expressing mammalian viral proteins, less stability of VLPs, costly product formation, and difficulties in the separation of morphologically similar contaminant proteins in different host systems Pattenden et al. Accordingly, a number of studies have attempted to address these problems.
In a study, Bundy et al. Several advantages were listed over the currently used cell-based systems, including the redirection of metabolic resources more toward in vitro transcription and translation, one-step purification as well as recovery, and the removal of the laborious procedures of cell transformation.
For enhancing the stability of VLPs, Bundy and Swartz controlled the redox potential of the reaction system, allowing them to be able to control the formation of disulfide bonds between capsid monomers, thus altering VLP stability. In their experiment, they produced azide and alkyne methionine analogs on the surface of the VLPs.
Their system produced 0. Further research and better optimization of CFPS protocols are needed to improve the robustness and potency of this technique when considering the creation of virus and VLPs. In the near future, CFPS systems may well replace the currently used cell-based methods at the production scale, given the advantages that the CFPS systems have over established methods.
Biocontainment is an aspect of biosafety concerning the organisms and species that can pose a risk to human health and ecology, and specifically covers their physical containment within secure areas, toward prohibiting their release into the wider community. Accordingly, there is a pressing need to develop new technologies to deal with biocontainment risks that threaten biosafety and biosecurity, and these technologies would ultimately serve to sharply decrease the potential severity and danger presented by genetically modified organisms.
Among the many newer strategies that have been proposed to date, a number of them that involve cell-free systems have been suggested Lee et al. The benefit of deriving proteins through CFPS systems in this way stems from their ability to remain abiotic, lacking most of the normal biotic processes of cells, while involving genes and DNA, which would be dividing, duplicating, and mutating.
In all cases, the use of CFPS systems meant that the experimental process exhibited lower biosafety risk than with living systems, which may often be an undervalued aspect of cell-free systems in general. This kind of xenobiological biosafety barrier is a deliberately sought out target for the field of xenobiology or xeno nucleic acids, and benefitting from use of cell-free systems underlines a clear and strong potential for these technologies to augment the biocontainment strategies of the future.
Synthetic biology is a modern and innovative scientific discipline with an aim to improve the existing industrial practices, addressing issues of poor yields and poor cost-to-product ratios, as well as the problems of current practices that inevitably damage our ecosystems through polluting acts. In any of these cases, it would be prudent to consider alternatives, and it is in synthetic biology that novel and alternative routes for the fabrication of many value-added products have been found with a compelling amount of accomplishments and an ever-fertile basis to grow future products and industries.
In this context, we have reviewed and discussed CFPS, covering its numerous successes achieved to date and the wide-reaching potential for it to develop, as well as some of the necessary steps required. For improving methods of protein production, CFPS systems have shown great efficiency to generate high levels of expression, purity, and yield, in addition to allowing the easier incorporation of labeled amino acids, factors that permit better NMR analysis of protein structures Morita et al.
The relative ease of working with CFPS systems means that the time-consuming and laborious processes of cloning can be minimized, meaning that large-scale libraries of functional proteins can be made easier than before Sawasaki et al. These findings indicate that CFPS systems could comfortably be expanded and used in the expression as well as testing of higher protein libraries designed for multi-well formats and experiments, allowing highly complex studies and high-throughput experiments that are assisted by this technology to work much faster that too at a lower cost, relative to current practices.
Accordingly, an area of further research would be to test the full potential of CFPS systems and their applicability to assist other types of complex and high-throughput experiments.
We have discussed a number of difficult proteins that have already been produced with CFPS systems, including several restriction endonucleases Goodsell, , human microtubule binding protein Betton, , and cytolethal distending toxin Ceelen et al.
The expression of this range of difficult proteins is highly inspiring that it could be translated toward other challenging proteins. The importance that protein and enzyme products play in modern medicine and in the biological studies cannot be understated, and accordingly, it follows that one component of the progression for this technology may simply be to apply it rigorously to the proteins that remain too difficult to study.
CFPS has the potential to drive new findings in various fields that could well revolutionize many medical treatments, as well as the biomedical studies of cancer, viral infections via human receptors , and antibiotic resistance, among many others.
The currently used cellular lysates are derived from E. It would be of great interest to use other sources of cell-free materials and substrates, especially toward addressing one of the problems in CFPS systems that concerns the post-translational modifications of products. These modifications include glycosylation, disulfide bonding, and correct protein folding. Further developments in CFPS systems would accordingly involve the testing and assay of new cellular lysates, toward identifying the best ones for all of the desired modifications possible, a project that could eventually develop into an in silico form as with numerous other protein synthesis and design assisting platforms.
Currently, cell-free systems are being used for cost-effective detection of Ebola, Zika, and dengue viral strains Pardee et al. The invention of cheaper and portable tests that employ cell-free systems can revolutionize the manner in which these lethal diseases can be detected and dealt with, reducing their burden on human health.
We believe that, in the future, CFPS will be able to address these diseases more sternly, as the damage that they do to people and communities is too high. Having realized these powerful methods for detecting these diseases, we appreciate the potential for how these can be adapted against the new viral outbreaks in future, to assist in better disease management than before.
This should also be expanded for the other diseases of the world that remain difficult to diagnose and treat, where ever appropriate.
In other areas, CFPS systems have been used in a wide range of experiments, including the production of proteins that incorporate toxic amino acids such as canavanine Worst et al. Many of these experiments give clear indications on the future work that must be performed for CFPS systems. In one avenue, cell-free TX—TL systems have allowed the incorporation of L-canavanine and L-hydroxy-lysine into proteins, opening the door for the future examination of these and other amino acid replacements.
The benefit here is that, with the expansion of basic language of proteins, they are now capable of possessing novel functionalities that are otherwise toxic to living cells, opening a whole new world for modified protein and enzyme functionalities that haven't been considered before. There is an exigency for more and better drugs against the myriad forms of cancer, and this result demonstrates that CFPS systems could be well-adapted for their synthesis or for improving the existing methods.
Viruses and VLPs can be used to develop experimental gene therapy treatments, drug delivery, diagnostic tools, and nanotechnology applications Shirbaghaee and Bolhassani, Within all of these aspects, the use of CFPS systems has enabled biologists to advance in each of these distinct areas, discovering new results and findings. In general, we believe that this range of studies that have been benefited by cell-free expression systems offer a very promising belief that these systems can be redeployed into many other scientific studies, offering advantages that permit countless other interesting and compelling experiments to be performed, more quickly and at a lower cost, similarly to the ones that we discussed.
Even in the smallest of cases, if the use of these systems can save money and time, it may well open the door for lowering the barriers to allow entry of many scientists and their projects, offering greater diversity of ideas and experiments to be possible. Lastly, we believe that the CFPS systems that we have discussed have already realized numerous successes and with the current rate that modern science and synthetic biology is growing, it is clear that novel developments and innovations must follow.
We must extrapolate these successes to address many existing world issues in novel, safer, more efficient, and greener ways, to benefit the health of the planet, and ultimately remove our reliance on non-renewable and polluting sources of valuable products and energy. VK and DB have proofread and given comments as well as suggestions. VS has supervised and finalized the manuscript.
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