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Proteins in and of themselves are relatively uncomplicated. However, the various substructures and elements that comprise proteins are intricate when considering chemical reactions and connectivity of specific structures and functions of proteins.

Proteins are organic compounds that contain four elements: nitrogen, carbon, hydrogen, and oxygen. To comprehend the full scope of proteins, it is crucial to understand various properties, including the basic biological molecule, peptides, polypeptide chains, amino acids, protein structures, and the processes of protein denaturation.

Polypeptides and Proteins: What's the difference

According to IUPAC, polypeptides with a molecular mass of 10,000 Da or more are classified as proteins. At times, the term 'proteins' refers to molecules with 50-100 combined amino acids. Each protein contains one or more polypeptide chain. The chemical properties and order of the amino acids determines the structure and function of the polypeptide. Each of these polypeptide chains is made up of amino acids, which are joined together in a detailed, deliberate and specific order.

What do proteins do for the body?

Proteins play vital roles in human biochemistry, primarily serving as the body's building blocks. They act as precursors to various biologically relevant molecules, and imbalances—either excess or deficiency of proteins—can lead to diseases, resulting in nervous system defects, metabolic issues, organ failure, and even death.

Proteins exhibit a diverse range of functions in the body, which can be categorized alphabetically:

Antibodies : These proteins bind to specific foreign particles, such as viruses and bacteria, contributing to the body's defense. Example: Immunoglobulin G (IgG).

Enzymes : Responsible for executing the multitude of chemical reactions within cells, enzymes also assist in the formation of new molecules by interpreting genetic information from DNA. Example: Phenylalanine hydroxylase.

Messenger Proteins : Some types of hormones fall into this category, transmitting signals to coordinate biological processes among different cells, tissues, and organs. Example: Growth hormone.

Structural Components : Providing structure and support for cells, these proteins also enable bodily movement on a larger scale. Example: Actin.

Transport/Storage : These proteins bind and carry atoms and small molecules within cells and throughout the body. Example: Ferritin.

As proteins are essential for cell and tissue growth, ensuring an adequate protein intake is particularly crucial during periods of rapid growth or increased demand, such as childhood, adolescence, pregnancy, and breastfeeding

Protein Sources

Protein-rich foods come from both animal and plant sources. Here are examples of high-protein foods. The values are given per 100 grams of the food:

Animal-Based Proteins : Chicken Breast (cooked, skinless): ~31 grams Salmon (cooked): ~25 grams Ground Beef (cooked, 90% lean): ~26 grams Eggs: ~13 grams Greek Yogurt: ~10 grams Milk: ~3.4 grams Cheese (cheddar): ~25 grams

Plant-Based Proteins : Lentils (cooked): ~9 grams Chickpeas (cooked): ~8 grams Black Beans (cooked): ~8 grams Quinoa (cooked): ~4 grams Tofu: ~8 grams Almonds: ~21 grams Peanut Butter: ~25 grams

While both animal and plant-based proteins provide essential amino acids, there are some distinctions:

1. Amino Acid Profile : Animal proteins generally contain all essential amino acids in the right proportions, making them "complete" proteins. Most plant proteins lack one or more essential amino acids, making them "incomplete." However, combining different plant protein sources throughout the day can provide a complete amino acid profile.

2. Digestibility : Animal proteins are often more easily digestible than some plant proteins. However, cooking and preparation methods can enhance the digestibility of plant proteins.

3. Nutrient Content : Animal products usually contain additional nutrients like vitamin B12, heme iron, and omega-3 fatty acids, which may be less abundant in plant sources.

Recommended Daily Protein Intake . The amount of protein a person needs can vary based on factors such as age, sex, physical activity, and overall health. However, a general guideline is: Adults : The Recommended Dietary Allowance (RDA) for protein is 0.8 grams per kilogram of body weight. For example, a sedentary adult weighing 70 kilograms would need around 56 grams of protein per day. Athletes and those engaging in heavy physical activity may require more.

It's important to note that individual protein needs can vary, and consulting with a healthcare professional or a registered dietitian can provide personalized recommendations based on specific health goals and conditions.

Protein Basics

Proteins are among the most abundant organic molecules in living systems and are vastly diverse in structure and function as compared to other classes of macromolecules. Even one cell contains thousands of proteins, which function in a variety of ways. Proteins are composed of 20 different amino acids, for which genomes dictate the specific amino acids and their sequences.

As a result, variances in protein activities and functions occur due to the complex structural and functional properties of long-evolved proteins. Such properties include: 1. The process of rapid protein folding. 2. Binding sites specific to small groups of molecules. 3. Balanced flexibility in structures and rigidity levels in order to maintain protein functions. 4. Congruent protein surface structures suitable for a protein's environment. 5. Protein vulnerabilities to degradation cause damage, thus rendering protein useless.

Several structural materials are contained within proteins that exist in all living organisms. The many diverse functions relative to these structures are: • Catalysis – Increases rates of chemical reactions within proteins (e.g. enzymes). • Structural – Strengthens bones, tendons and skin (e.g. collagen). • Movement – Muscle contractions permit movement (e.g. myosin). • Metabolic regulation – Joining or taking biomolecules that are needed by the cell. • Transport – Moves oxygen throughout the body (e.g. hemoglobin). • Defense – Protein antibodies serve as protection against foreign pathogens (e.g. immunoglobulin).

Proteins form in different shapes and structures. Some proteins are globular (spherical), compact and water-soluble, while other proteins are fibrous and elongated, physically tough, and water-insoluble. Chemical bonds are responsible for the shapes that proteins maintain.

The 4 Protein Structures

Four different structures of protein serve to influence specific protein activities. Different sequences of amino acids form different shapes and thus, different proteins. All protein activity is based on these four structures:

Primary Structure : The primary structure of a protein is its amino acid sequence. The base (repeat) sequence of the gene codes are comprised of three amino acids (glycine; proline; «x» - any other amino acid). This sequence of amino acids bonded together creates a polypeptide (poly = many) bond, or chain. The primary structure of a protein is linear .

Secondary Structure : The secondary structure takes the chains (primary) and folds, or coils them. These parts attract to one another to form structures that have α(alpha)-helices and β(beta)-pleated sheets. These form as a result of hydrogen bonds between the peptide groups of the main (primary) chain. These secondary-structure proteins contain regions that are cylindrical (α-helices, spiral shape) and/or regions that are planar (β-pleated sheets, ribbon with peaks and valleys). The secondary structure of a structure is three dimensional .

Tertiary Structure : The tertiary structure of a protein is its three-dimensional conformation that is created when the protein folds. Hydrogen bonds stabilize the folding occurrences. Other intramolecular bonds that stabilize the folding processes include hydrophobic interactions; ionic bonds; and disulphide bridges. These bonds are formed between the R groups of amino acids. They contain the nonpolar parts of proteins which result in attractions and repulsions and become coiled up in one area, creating a very complex structure. The tertiary structure is the overall shape of the protein for which most are globular in shape, or fibrous – long and thin.

Quaternary Structure : A quaternary structure is formed when two or more tertiary polypeptide chains form a single or full protein. Certain proteins may have a non-polypeptide structure, thus belonging to a prosthetic group, while other proteins are conjugated. Here unique patterns are formed via hydrogen bonding.

Chemical Bonding in Proteins

The amino acids of a polypeptide are linked together in chains by their neighboring covalent bonds ( peptide bonds ). In turn, each bond forms in a dehydration synthesis (condensation) reaction. Throughout the protein-synthesis process, the carboxyl group of the amino acid reacts with the amino group of an incoming amino acid, releasing a molecule of water. This process occurs at the end of the growing polypeptide chain, thus resulting in a bond between amino acids, known as a peptide (or polypeptide) chain .

A polypeptide chain has two ends, which are chemically distinct from each other. This is due to the amino acids, which produce directionality of the chain. The two ends of the polypeptide chain are comprised of: • A free amino group, which is the amino terminus (N-terminus). • A free carboxyl group (C-terminus). • Each terminus is located at the opposite end of the other.

In addition to the polypeptide bonds, sulfide bridge formations and Schiff base formations are two other chemical reactions that can take place during cell activities that are «directed» by amino acids, which comprise the basis of all proteins.

The amino acids' interactions cause a protein to fold into its mature shape (tertiary). Such folding happens because of the rotation of bonds within the amino acids, as well as bonds joining various amino acids.

Peptide bonds form between the carbonyl carbon and the nitrogen, which is another peptide bond. Nitrogen bonds to carbonyl carbon, then to the peptide linkage, back to the nitrogen, and so on. From the chains, the backbones are interacting. Furthermore, nitrogen is electro-negative such that it consumes electrons from the nitrogen. Here the hydrogen has a partially positive charge.

Conversely, oxygen is electro-negative. Oxygen takes electrons from the carbon which gives oxygen a partially negative charge. Simply put, the hydrogen and the oxygen are attracted to each other, creating a hydrogen bond . From there, these two connected chains form a β-pleated sheet . The backbones then interact with each other and move in the same direction, which is known as a parallel β-pleated sheet .

The β-pleated sheet takes on a certain pattern, moving in the order of nitrogen, α carbon, and then carbonyl carbon. This is on the left side of the protein. On the right side, the pattern moves in the order of carbonyl carbon, α carbon, and then nitrogen. Note that these processes are traveling in opposite directions with the hydrogen bonds between these partially positive ends of the nitrogen-hydrogen bonds remaining at the hydrogen end. The hydrogen bonds and the backbones are still parallel, but again, they are moving in different directions. This is called an anti-parallel β-pleated sheet , which is another form of a secondary structure.

The backbone of the protein can also become a helical structure (also a secondary structure) where the hydrogen bonds form between the different layers of the helix. Remember that oxygen has a partially negative charge, and that hydrogen has a partially positive charge. At any rate, the resulting hydrogen bond gives the protein its helical structure. So these interactions highlight the in-depth and interrelational processes that create the structure, dynamics, and the functions of proteins.

All proteins have the above-mentioned processes in common, but not all proteins possess the more complicated and interactive tertiary and quaternary structures . Several molecular interactions and thermodynamic changes can transpire within these highly complex molecular structures of protein.

Protein Denaturation

Protein denaturation occurs when the three-dimensional structures (in secondary, tertiary, or sometimes quaternary structures) of proteins is altered. Denaturation makes protein non-functional, or at least unable to perform its usual functions. However, the peptide chains are left intact following denaturation, as are the chemical properties of protein. Most usually, the denaturation process is irreversible.

Factors such as heat, cold, or unfavorable pH can cause protein denaturation resulting in damage between the molecular structures of the peptide bonds, thus breaking the otherwise covalent bonds. «Healthy» proteins fold over one another, but denaturation causes them to unravel.

To understand how factors such as high temperature and high or low pH cause denaturation, it is important to remember the fact that hydrogen bonds are crucial in the secondary, tertiary, and sometimes the quaternary structures of protein. A hydrogen bond is a type of peptide bond. Carbon, oxygen, and nitrogen are equally essential in the aforementioned protein structures.

Too much heat, for example, will result in the twisting, bending and rotating of functional molecular groups, and since hydrogen is known to be fragile, it is more vulnerable to ruptures. Temperature increases disrupt the existing hydrogen bonds as well as other non-polar hydrophobic interactions. As the temperature increases, so does the kinetic energy. This causes the molecular components of the protein to vibrate, which leads to the broken bonds. This, in turn, creates a pattern of ruptures or breaks of any nearby hydrogen bonds.

References 1. Hegyi H, Bork P. On the classification and evolution of protein modules. J Protein Chem. 1997 Jul;16(5):545-51 . [ PubMed ]. 2. A. D. Mclachlan. Protein Structure and Function. Annu. Rev. Phys. Chem. 1972.23:165-192 . 3. Jay R. Hoffman, Michael J. Falvo. Protein - Which is Best? J Sports Sci Med. 2004 Sep; 3(3): 118-130 . [ PubMed ]. 4. Kent SB. Total chemical synthesis of proteins. Chem Soc Rev. 2009 Feb;38(2):338-51 . [ PubMed ].

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Ribosomes, Transcription, and Translation

The genetic information stored in DNA is a living archive of instructions that cells use to accomplish the functions of life. Inside each cell, catalysts seek out the appropriate information from this archive and use it to build new proteins — proteins that make up the structures of the cell, run the biochemical reactions in the cell, and are sometimes manufactured for export. Although all of the cells that make up a multicellular organism contain identical genetic information, functionally different cells within the organism use different sets of catalysts to express only specific portions of these instructions to accomplish the functions of life.

How Is Genetic Information Passed on in Dividing Cells?

When a cell divides, it creates one copy of its genetic information — in the form of DNA molecules — for each of the two resulting daughter cells. The accuracy of these copies determines the health and inherited features of the nascent cells, so it is essential that the process of DNA replication be as accurate as possible (Figure 1).

One factor that helps ensure precise replication is the double-helical structure of DNA itself. In particular, the two strands of the DNA double helix are made up of combinations of molecules called nucleotides . DNA is constructed from just four different nucleotides — adenine (A), thymine (T), cytosine (C), and guanine (G) — each of which is named for the nitrogenous base it contains. Moreover, the nucleotides that form one strand of the DNA double helix always bond with the nucleotides in the other strand according to a pattern known as complementary base-pairing — specifically, A always pairs with T, and C always pairs with G (Figure 2). Thus, during cell division, the paired strands unravel and each strand serves as the template for synthesis of a new complementary strand.

What Are the Initial Steps in Accessing Genetic Information?

RNA molecules differ from DNA molecules in several important ways: They are single stranded rather than double stranded; their sugar component is a ribose rather than a deoxyribose; and they include uracil (U) nucleotides rather than thymine (T) nucleotides (Figure 4). Also, because they are single strands, RNA molecules don't form helices; rather, they fold into complex structures that are stabilized by internal complementary base-pairing.

mRNA is the most variable class of RNA, and there are literally thousands of different mRNA molecules present in a cell at any given time. Some mRNA molecules are abundant, numbering in the hundreds or thousands, as is often true of transcripts encoding structural proteins. Other mRNAs are quite rare, with perhaps only a single copy present, as is sometimes the case for transcripts that encode signaling proteins. mRNAs also vary in how long-lived they are. In eukaryotes, transcripts for structural proteins may remain intact for over ten hours, whereas transcripts for signaling proteins may be degraded in less than ten minutes.

Cells can be characterized by the spectrum of mRNA molecules present within them; this spectrum is called the transcriptome . Whereas each cell in a multicellular organism carries the same DNA or genome, its transcriptome varies widely according to cell type and function. For instance, the insulin-producing cells of the pancreas contain transcripts for insulin, but bone cells do not. Even though bone cells carry the gene for insulin, this gene is not transcribed. Therefore, the transcriptome functions as a kind of catalog of all of the genes that are being expressed in a cell at a particular point in time.

What Is the Function of Ribosomes?

Ribosomes are complexes of rRNA molecules and proteins, and they can be observed in electron micrographs of cells. Sometimes, ribosomes are visible as clusters, called polyribosomes. In eukaryotes (but not in prokaryotes), some of the ribosomes are attached to internal membranes, where they synthesize the proteins that will later reside in those membranes, or are destined for secretion (Figure 6). Although only a few rRNA molecules are present in each ribosome, these molecules make up about half of the ribosomal mass. The remaining mass consists of a number of proteins — nearly 60 in prokaryotic cells and over 80 in eukaryotic cells.

Within the ribosome, the rRNA molecules direct the catalytic steps of protein synthesis — the stitching together of amino acids to make a protein molecule. In fact, rRNA is sometimes called a ribozyme or catalytic RNA to reflect this function.

How Does the Whole Process Result in New Proteins?

After the transcription of DNA to mRNA is complete, translation — or the reading of these mRNAs to make proteins — begins. Recall that mRNA molecules are single stranded, and the order of their bases — A, U, C, and G — is complementary to that in specific portions of the cell's DNA. Each mRNA dictates the order in which amino acids should be added to a growing protein as it is synthesized. In fact, every amino acid is represented by a three-nucleotide sequence or codon along the mRNA molecule. For example, AGC is the mRNA codon for the amino acid serine, and UAA is a signal to stop translating a protein — also called the stop codon (Figure 7).

Molecules of tRNA are responsible for matching amino acids with the appropriate codons in mRNA. 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).

In prokaryotic cells, transcription (DNA to mRNA) and translation (mRNA to protein) are so closely linked that translation usually begins before transcription is complete. 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.

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Biochemistry, protein synthesis.

Jacob E. Hoerter ; Steven R. Ellis .

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Last Update: July 17, 2023 .

Introduction

Our understanding of each of the biological sciences becomes heightened by the study of biochemistry and molecular biology. In the last few decades, advances in laboratory techniques for the study of these microscopic sciences have led us to a greater understanding of the central dogma of molecular biology – that DNA transcribes RNA which then gets translated into protein. Understanding protein synthesis is paramount in studying various medical fields, from the molecular basis of genetic diseases through antibiotic development to expressing recombinant proteins as drugs or clinical laboratory reagents. As one of the foundational concepts in biology, protein synthesis is sufficiently complex that many believe it evolved once, giving the protein synthetic machinery in all organisms on the planet a common ancestry. Despite having certain underlying similarities in their mechanism, protein synthesis in the three major lines of descent (bacteria, archaea, and eukaryotes) has diverged to the point that substantive mechanistic differences have arisen. These differences have been exploited in nature as organisms produce compounds targeting the protein synthetic machinery of competitors as they vie for limited resources. Science has modified many of these compounds that target the machinery for protein synthesis in pathogenic microbes for use in the clinic as antibiotics. As our understanding of the mechanisms of protein synthesis continues to grow, there will likely be countless additional applications for this knowledge in medicine, research, and industry.

Fundamentals

Protein synthesis involves a complex interplay of many macromolecules.

The eukaryotic ribosome has two subunits: a 40S small subunit and a 60S large subunit. Together, the eukaryotic ribosome is 80S. There are several sites of functional significance, but the most important ones are the A (aminoacyl), P (peptidyl), and E (exit) sites. The eukaryotic ribosome is a ribonucleoprotein complex composed of 4 RNAs and 80 proteins. Many of the functions of the ribosome, including catalyzing peptide bond formation, are attributed to ribosomal RNA (rRNA) rather than ribosomal proteins, which instead play a primary role in subunit assembly. Ribosomes can be found either adherent to membranes of the endoplasmic reticulum or free within the cytoplasm. [1]
Bacterial ribosomes have two subunits, 30S and 50S, that join to form a 70S particle. In general, bacterial ribosomes are smaller than their eukaryotic counterparts, including fewer ribosomal proteins (55) and shorter rRNAs (3 in total). Certain regions of rRNA and some of the ribosomal proteins remain conserved between bacteria and eukaryotes. Other regions of rRNA and proteins are unique to either eukaryotes or bacteria and account in part for differences in mechanisms of protein synthesis discussed above.
Eukaryotic cells contain a second type of ribosome found within the mitochondrion, which maintains a system of protein synthesis distinct from that found in the cytoplasm. Despite their presence in eukaryotic cells, the origins of the mitochondrial ribosome are traceable to bacteria, consistent with the endosymbiont theory of mitochondrial origins. Care must be taken during antibiotic development to avoid targeting characteristics of the mitochondrial ribosome shared with bacterial ribosomes.
Messenger RNA (mRNA): the mRNA is another type of ribonucleic acid that functions to carry the coding section of a gene for protein synthesis. It contains portions of non-coding and coding sequences. The coding sequence groups nucleotides into codons, which are three specific nucleotides that correspond to a particular amino acid specified by the genetic code. [2]
Transfer RNA (tRNA): tRNAs are adaptors bridging the nucleotide sequence found in mRNAs to the amino acid sequence found in a growing protein. Transfer RNAs assume a cloverleaf-like secondary structure with an amino acid linked to its 3’ end through an ester linkage and a stretch of three nucleotides at the base of the cloverleaf referred to as the anticodon. The three bases of the anticodon base pair with complementary codon sequences in an mRNA during the process of protein synthesis. This base-pairing interaction plays a critical role in the readout of the genetic code from mRNA to protein. There are 20 different aminoacyl-tRNA synthetases, one for each of the 20 common amino acids. Once an amino acid links to its cognate tRNA it is referred to as an aminoacyl tRNA, or “charged” tRNA. [3]
Genetic code: The genetic code sequence is three nucleotides originally encoded an organism’s genome that specifies individual amino acids found in proteins. There are 20 common amino acids used by the protein synthetic machinery and 64 potential sequence permutations of the four bases used to specify the 20 amino acids. Early studies revealed that the code was degenerate, with many of the amino acids specified by multiple 3-base combinations. In general, when multiple codons specify a single amino acid, degeneracy is found at the third or “wobble” position. [1] Sixty-one of the 64 sequence permutations specify amino acids, whereas three of the sequence permutations serve as “stop” codons to terminate protein synthesis. While initially thought to be the same for all living organisms, scientists now know that there are a small number of deviations from the universal code found in mitochondria and specific bacterial species.
Genetic code and human disease: Appropriate readout of the genetic code is essential for human health. Mutations that alter protein-coding sequences can affect proteins in many different ways. The effect of mutations on the coding sequence can classify as either synonymous or nonsynonymous depending on whether they are predicted to alter the primary structure of a protein.
Synonymous mutations relate to the degeneracy of the code and the fact that changes in base sequence may not have an effect on which amino acid a codon represents (though it should be noted that some synonymous mutations may affect pre-mRNA splicing and so influence a protein’s primary structure). Synonymous mutations typically fall in the third position of a codon.
Nonsynonymous mutations fall into three different classes:
Missense mutations where there is substitution of one amino acid for another.
Nonsense mutations which introduce a premature termination codon in an mRNA sequence. These mutations typically result in a truncated protein.
Frameshift mutations result from insertion or deletion mutations that shift the reading frame of a coding sequence such that sequencing downstream of the mutational event no longer code for the correct amino acid sequence of a protein.
Protein factors– the process of protein synthesis requires multiple non-ribosomal proteins that transiently participate during the initiation, elongation, and termination phases of protein synthesis. These factors are named for the phase in which they function (for example, eukaryotic initiation factor 2, eIF2). [2]
Issues of Concern

The proteins that a cell expresses are the ultimate manifestation of its phenotype. Cells within tissues of the human body have variable phenotypic expression involved in defining tissue organization and function despite having identical genomes due to the differential expression of genes within the genome. While the differential regulation of gene expression primarily occurs at the level of transcription, regulation of gene expression can also take place at the post-transcriptional level, including regulated translation. Because of the importance of protein expression to the phenotypic properties of a cell, errors in the cellular proteome manifested at all levels of the correct readout of genetic information from gene to protein can have broad implications on health.

Cellular Level

The eukaryotic cell is compartmentalized, with different cellular compartments defined by biological membranes. The synthesis of components of the translational machinery begins with the transcription of mRNAs, tRNAs, and rRNAs in the nucleus by RNA polymerases II, III, and I, respectively. Transfer RNAs and the mRNAs encoding ribosomal proteins exit the nucleus and the latter get translated in the cytoplasm. Ribosomal proteins then return to the nucleus where they assemble hierarchically on rRNAs being transcribed by RNA polymerase I. This assembly process defines a compartment of nucleus referred to as the nucleolus. Ribosome assembly is a complex process involving hundreds of accessory factors that transiently associate with ribosomal subunits during their maturation. While most of the steps involved in maturing ribosomal subunits occur within the nucleolus before the subunits exiting through nuclear pores, final steps in subunit maturation occur in the cytoplasm. Ribosomes translating most cellular mRNAs do so as free ribosomes in the cytoplasm. In contrast, ribosomes translating mRNAs encoding proteins destined for secretion from the cell or resident proteins of the endoplasmic reticulum, Golgi apparatus, lysosome, or plasma membrane get localized to the endoplasmic reticulum membrane. [4]

Briefly, translation can be broken down into three phases initiation, elongation, and termination. Initiation consists of identifying the exact site in the sequence of nucleotides in an mRNA to begin translation. This process has significant differences between eukaryotes (described here) and prokaryotes. Upon identification of the start site for translation, elongation ensues as the ribosome moves along the mRNA “reading” groups of three nucleotides that specify each amino acid added to the growing polypeptide chain. Finally, termination occurs when the ribosome encounters one of three termination codons, and the completed protein gets released from the ribosome.

Translation begins with the assembly of an 80S initiation complex on mRNA. This process involves identifying appropriate codon to initiate translation. The AUG codon specifies the amino acid methionine and virtually all proteins specified by the genetic code begin with methionine. In eukaryotes, the AUG used to initiate protein synthesis is usually the first AUG downstream of the cap structure, found at the 5’ end of the mRNA. A protein complex known as eIF4F recognizes the cap structure. The eIF4F complex then recruits the 43S pre-initiation complex comprised of 40S subunits together with a ternary complex formed of the initiator tRNA (Met-tRNA), eIF2, and GTP to the 5’ end of an mRNA. The 40S complex subsequently scans down the mRNA until encountering the first AUG and the 48S initiation complex forms. In addition to eIF4F and eIF2, multiple other initiation factors facilitate the formation of the 48S initiation complex. At this point, the 60S ribosomal subunit joins the 48S initiation complex, all initiation factors are released, and the elongation phase of translation is set to begin. In the 80S initiation complex, the initiator Met-tRNA is base-paired to the initiating AUG in the ribosomal P site with the next codon of the mRNA positioned in the ribosomal A site. Translational re-initiation facilitation occurs by the interaction of the eIF4F complex with both the 5’ cap and the 3’ polyA tail of an mRNA. [5]

As with initiation, elongation requires the use of non-ribosomal proteins known as elongation factors. Eukaryotic EF1A (eEF1A) forms ternary complexes with aminoacyl-tRNAs and GTP. These ternary complexes enter the empty A site of the ribosome and if an appropriate codon-anticodon interaction forms between the incoming aminoacyl-tRNA and the codon in the A site, GTP will be hydrolyzed and eEF1A released. At this point, the peptidyl-transferase site of the ribosome catalyzes peptide bond formation as the free amino group of the incoming aminoacyl-tRNA attacks the ester bond linking the growing polypeptide to the tRNA in the ribosomal P site. The resultant uncharged tRNA occupying the P site moves to the E (exit) site and leaves the ribosome. The growing polypeptide chain previously in the P site is now elongated by one amino acid as it transfers to the aminoacyl-tRNA in the A site. The peptidyl-tRNA in the A site is then translocated to back to the P site with the help of eEF2 and GTP. The A site is now empty, and the entire process is repeated over and over again as the ribosome moves down the mRNA.

Termination occurs when eRF1, a release factor structurally analogous to tRNA, recognizes termination codons in an mRNA and recruits eRF3 to hydrolyze the polypeptide chain from the tRNA occupying the P site. Termination of translation completes by the dissociation of the ribosomal subunits, which are now capable of initiating another round of protein synthesis. Multiple ribosomes can translate a single mRNA simultaneously forming complexes known as polysomes. [5] [6] [7]

There are many possible methods of confirming that a particular protein is being synthesized.

Immunostaining

Because of the large number of proteins synthesized in a typical cell, verifying the presence of a particular protein is understandably challenging. One way to confirm the presence of a specific protein in a clinical specimen is through immunostaining. This technique introduces an antibody to a protein of interest, and the exquisite specificity of the antibody serves for protein detection.

In immunostaining, the specimen is incubated with a primary antibody solution. This antibody can contain a fluorescent molecule on its heavy chain or an enzyme (such as horseradish peroxidase) that will fluoresce in the presence of a suitable substrate. The light released can be visualized under a microscope or exposed to photosensitive film in a dark room for later development. Immunostaining can either be direct where the primary antibody possesses the means of fluorescent detection or indirect, where a secondary antibody raised against the primary antibody is detectable via fluorescence. [8]

Protein Electrophoresis

As with nucleic acids, proteins can be separated based on size and/or charge using gel electrophoresis. Proteins can be run in their native configurations or undergo denaturing before electrophoresis. In denaturing electrophoresis, a detergent such as sodium dodecyl sulfate (SDS) is used to disrupt non-covalent bonding forces within proteins. SDS also gives proteins common charge to mass ratios, so the only force operating during SDS-polyacrylamide gel electrophoresis is the molecular sieving action of the polyacrylamide gel. Proteins separated in this manner can be detected either non-specifically with dyes like coomassie blue or specifically using antibodies in a procedure referred to as Western blotting or immunoblotting.

Pathophysiology

Many human diseases result from changes in protein sequence caused by mutations that alter the correct readout of genetic information from gene to a functional protein. Defects in the protein synthetic machinery also cause a small but growing number of human diseases. Examples of such pathologies follow.

Sickle Cell Anemia

Human hemoglobin contains two alpha and two beta chains to create a heterotetramer. In Sickle Cell Anemia, the sixth codon of the beta chain contains a missense mutation, in which glutamic acid, a charged amino acid, is replaced with valine, a neutral amino acid. This single amino acid difference affects the tertiary and quaternary structures of hemoglobin such that it distorts the biconcave shape of erythrocytes into sickle shapes in certain conditions. [9]

Duchenne Muscular Dystrophy

Like many X-linked diseases, DMD primarily affects males at an early age. It is characterized clinically by muscle weakness, calf pseudohypertrophy, and the Gower sign in a child. One of the pathophysiologic origins of this disease is the formation of a premature stop codon in an early exon of the dystrophin gene, which leads to a truncated dystrophin protein which compromises the integrity of the sarcomere and contractile function of the muscle. [10]

Diamond-Blackfan Anemia

While many human diseases result from mutations in the coding sequences of genes that affect protein production, Diamond-Blackfan anemia (DBA) is one of a growing number of conditions resulting from defects in the protein synthetic machinery. DBA is caused by autosomal dominant mutations in genes encoding proteins of either the 40S or 60S ribosomal subunit. While the exact mechanisms underlying the pathophysiology of DBA are currently unknown, it seems likely that changes in cellular proteomes (the protein composition of a cell) resulting from suboptimal numbers of ribosomes contribute in part to the clinical features of the disease. These clinical features include a deficit in red blood cell production, small size, and a heterogeneous number of congenital anomalies. [11]

Clinical Significance

The clinical significance of protein synthesis lies not only in human translation but in differences between human and bacterial translation. The bacterial ribosome (70S) has the same core components and many structurally similar sites compared to the eukaryotic ribosome (80S). However, translational differences between humans and bacteria create targets for antimicrobial drugs. These differences allow certain antibiotics to bind selectively to bacterial ribosomes at low concentrations, targeting bacteria selectively and either inhibiting growth or killing the microbe. Several commonly prescribed antibiotics target specific components of the bacterial ribosome and mRNA. Aminoglycosides target the 30S small ribosomal subunit; specifically, this class binds to the rRNA segment active in the A site. The tetracyclines operate similarly by competing for the A site with charged aminoacyl tRNA. The macrolide antibiotics act on the 50S large ribosomal subunit. When they bind to the rRNA of the large subunit, it prevents the formation of the peptide bond and promotes the early expulsion of the tRNA in the P site. [12] [3]

The clinical manifestations of differences in protein synthesis can also be useful in diagnosis. Native protein electrophoresis can help identify hemoglobinopathies in newborn screenings. Similarly, serum protein electrophoresis can identify characteristic M protein spikes of monoclonal protein expression in multiple myeloma.

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Disclosure: Jacob Hoerter declares no relevant financial relationships with ineligible companies.

Disclosure: Steven Ellis declares no relevant financial relationships with ineligible companies.

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Cite this Page Hoerter JE, Ellis SR. Biochemistry, Protein Synthesis. [Updated 2023 Jul 17]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-.

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Protein synthesis

Protein synthesis n., plural: protein syntheses Definition: the creation of protein.

Table of Contents

Protein synthesis is the process of creating protein molecules. In biological systems, it involves amino acid synthesis, transcription, translation, and post-translational events. In amino acid synthesis , there is a set of biochemical processes that produce amino acids from carbon sources like glucose .

Not all amino acids are produced by the body; other amino acids are obtained from the diet . Within the cells, proteins are generated involving transcription and translation processes. In brief, transcription is the process by which the mRNA template is transcribed from DNA.

The template is used for the succeeding step, translation. In translation, the amino acids are linked together in a particular order based on the genetic code. After translation, the newly formed protein undergoes further processing, such as proteolysis, post-translational modification, and protein folding.

Proteins are made up of amino acids that are arrainged in orderly fashion. Discover how the cell organizes protein synthesis with the help of the RNAs. You’re more than welcome to join us in our Forum discussion: What does mRNA do in protein synthesis?

Protein Synthesis Definition

Protein synthesis is the creation of proteins. In biological systems, it is carried out inside the cell. In prokaryotes , it occurs in the cytoplasm . In eukaryotes , it initially occurs in the nucleus to create a transcript ( mRNA ) of the coding region of the DNA . The transcript leaves the nucleus and reaches the ribosomes for translation into a protein molecule with a specific sequence of amino acids .

Protein synthesis is the creation of proteins by cells that uses DNA , RNA , and various enzymes . It generally includes transcription , translation , and post-translational events, such as protein folding, modifications, and proteolysis.

The term protein came from Late Greek prōteios , prōtos , meaning “first”. The word synthesis came from Greek sunthesis , from suntithenai , meaning “to put together”. Variant : protein biosynthesis.

Forum Question: Where does protein synthesis take place? Best Answer!

Prokaryotic vs. Eukaryotic Protein Synthesis

Proteins are a major type of biomolecule that all living things require to thrive. Both prokaryotes and eukaryotes produce various proteins for multifarious processes and functions. Some proteins are used for structural purposes while others act as catalysts for biochemical reactions.

Prokaryotic and eukaryotic protein syntheses have distinct differences. For instance, protein synthesis in prokaryotes occurs in the cytoplasm. In eukaryotes, the first step (transcription) occurs in the nucleus. When the transcript (mRNA) is formed, it proceeds to the cytoplasm where ribosomes are located.

Here, the mRNA is translated into an amino acid chain. In the table below, differences between prokaryotic and eukaryotic protein syntheses are shown.

Genetic Code

In biology, a codon refers to the trinucleotides that specify for a particular amino acid. For example, Guanine-Cytosine-Cytosine (GCC) codes for the amino acid alanine .

The Guanine-Uracil-Uracil (GUU) codes for valine. Uracil-Adenine-Adenine (UAA) is a stop codon. The codon of the mRNA complements the trinucleotide (called anticodon) in the tRNA.

What is the Genetic Code? “The genetic code is the system that combines different components of protein synthesis, like DNA, mRNA, tRNA…” More FAQ answered by our biology expert in the Forum: What does mRNA do in protein synthesis? Come join us now!

mRNA, tRNA, and rRNA

mRNA , tRNA , and rRNA are the three major types of RNA involved in protein synthesis. The mRNA (or messenger RNA) carries the code for making a protein. In eukaryotes, it is formed inside the nucleus and consists of a 5′ cap, 5’UTR region, coding region, 3’UTR region, and poly(A) tail. The copy of a DNA segment for gene expression is located in its coding region. It begins with a start codon at 5’end and a stop codon at the 3′ end.

tRNA (or transfer RNA), as the name implies, transfers the specific amino acid to the ribosome to be added to the growing chain of amino acid. 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 ). A peptidyl-tRNA is the tRNA that holds the growing polypeptide chain.

Unlike the first two, rRNA (or ribosomal RNA) does not carry genetic information. 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. rRNA has three binding sites: A, P, and E sites. 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.

Protein Biosynthesis Steps

Major steps of protein biosynthesis:

Transcription
Translation
Post-translation

Transcription is the process by which an 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. Similar to DNA replication, the transcription proceeds in the 5′ → 3′ direction. 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.

Step 1: Initiation

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 transcription start site in the transcription bubble binds to the RNA polymerase, particularly to the latter’s initiating NTP and an extending NTP . A phase of abortive cycles of synthesis occurs resulting in the release of short mRNA transcripts (about 2 to 15 nucleotides).

Step 2: Promoter escape

The next step is for the RNA polymerase to escape the promoter so that it can enter into the elongation step.

Step 3: Elongation

During elongation, RNA polymerase traverses the template strand of the DNA and base pairs with the nucleotides on the template (noncoding) strand. This results in an mRNA transcript containing a copy of the coding strand of DNA, except for thymines that are replaced by uracils. The sugar-phosphate backbone forms through RNA polymerase.

Step 4: Termination

The last step is termination. During this phase, the hydrogen bonds of the RNA-DNA helix break. In eukaryotes, the mRNA transcript goes through further processing. It goes through polyadenylation , capping , and splicing .

Translation is the process in which amino acids are linked together in a specific order according to the rules specified by the genetic code. It occurs in the cytoplasm where the ribosomes are located. It consists of four phases:

Activation (the amino acid is covalently bonded to the tRNA ),
Initiation (the small subunit of the ribosome binds to 5′ end of mRNA with the help of initiation factors)
Elongation (the next aminoacyl-tRNA in line binds to the ribosome along with GTP and an elongation factor)
Termination (the A site of the ribosome faces a stop codon)

Post-translation Events

Following protein synthesis are events such as proteolysis and protein folding . Proteolysis refers to the cleavage of proteins by proteases. Through it, N-terminal, C-terminal, or the internal amino-acid residues are removed from the polypeptide.

Post-translational modification refers to the enzymatic processing of a polypeptide chain following translation and peptide bond formation. The ends and the side chains of the polypeptide may be modified in order to ensure proper cellular localization and function. Protein folding is the folding of the polypeptide chains to assume secondary and tertiary structures.

Watch this video about Protein Translation:

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