Protein Synthesis (Translation)- Definition, Enzymes, Steps, Inhibitors
Protein synthesis is a process of creating protein molecules that are essential for life. Proteins are complex molecules that perform various functions in the cells, such as catalyzing chemical reactions, transporting substances, providing structure, regulating gene expression, and defending against pathogens. Proteins are made of smaller units called amino acids, which are linked together by peptide bonds to form long chains called polypeptides. The sequence of amino acids in a polypeptide determines the shape and function of the protein.
Protein synthesis is based on the genetic information encoded in the DNA of the cell. DNA is a double-stranded molecule that stores the instructions for making proteins in the form of nucleotide sequences. Each nucleotide consists of a nitrogenous base (adenine, thymine, cytosine, or guanine), a sugar (deoxyribose), and a phosphate group. The bases on one strand of DNA pair with the complementary bases on the other strand, forming a double helix structure. The order of bases on one strand of DNA specifies the order of amino acids in a protein.
However, DNA cannot directly interact with the protein-making machinery of the cell. Therefore, an intermediate molecule called messenger RNA (mRNA) is needed to transfer the genetic information from DNA to the site of protein synthesis. mRNA is a single-stranded molecule that is synthesized from a DNA template by an enzyme called RNA polymerase. mRNA has a similar structure to DNA, except that it has a different sugar (ribose) and a different base (uracil instead of thymine). The process of making mRNA from DNA is called transcription.
The site of protein synthesis is the ribosome, a complex structure composed of ribosomal RNA (rRNA) and proteins. Ribosomes are found either free in the cytoplasm or attached to the endoplasmic reticulum (ER), a membrane-bound organelle. Ribosomes have two subunits: a large subunit and a small subunit. The large subunit has three binding sites for another type of RNA called transfer RNA (tRNA). tRNA is a small molecule that carries an amino acid at one end and has an anticodon at the other end. The anticodon is a triplet of bases that is complementary to a codon, a triplet of bases on mRNA that specifies an amino acid. The process of making proteins from mRNA and tRNA is called translation.
Protein synthesis involves three main steps: initiation, elongation, and termination. Initiation is the start of translation, where the small ribosomal subunit binds to the 5` end of mRNA and scans for the start codon (usually AUG). Then, the initiator tRNA carrying methionine binds to the start codon and forms a complex with the large ribosomal subunit. Elongation is the continuation of translation, where successive tRNAs carrying different amino acids bind to the codons on mRNA and form peptide bonds with each other. The ribosome moves along the mRNA from 5` to 3` direction, adding amino acids to the growing polypeptide chain. Termination is the end of translation, where a stop codon (UAA, UAG, or UGA) on mRNA signals the release of the polypeptide and the dissociation of the ribosome.
Protein synthesis can vary slightly between prokaryotes (organisms without a nucleus) and eukaryotes (organisms with a nucleus). For example, prokaryotes have different types of ribosomes and initiation factors than eukaryotes. Prokaryotes can also initiate translation before transcription is completed, while eukaryotes have to transport mRNA from the nucleus to the cytoplasm before translation can begin.
Protein synthesis can be regulated at different levels by various factors, such as hormones, growth factors, stress signals, and environmental cues. Protein synthesis can also be inhibited by certain drugs or toxins that interfere with transcription or translation. Some examples of protein synthesis inhibitors are puromycin, streptomycin, chloramphenicol, erythromycin, cycloheximide, and diphtheria toxin.
Protein synthesis is a vital process for all living organisms that enables them to produce proteins according to their genetic code. Proteins are responsible for many biological functions and characteristics that define life.
Protein synthesis, or translation of mRNA into protein, occurs with the help of several molecules and macromolecules that contribute to the process. The protein synthesis machinery includes:
- Ribosomes: These are complex structures composed of ribosomal RNA (rRNA) and proteins, and are responsible for catalyzing the formation of peptide bonds between amino acids. Ribosomes have two subunits, a large one and a small one, that come together during translation. The large subunit has three active sites (A, P, and E) where tRNAs bind and interact. The small subunit has a binding site for mRNA. Ribosomes can be found in the cytoplasm, on the rough endoplasmic reticulum, or in mitochondria and chloroplasts .
- mRNA: This is the template that carries the genetic information from DNA to the ribosome. mRNA has a sequence of codons, each consisting of three nucleotides, that specify the amino acids to be added to the growing polypeptide chain. mRNA also has a 5` cap and a 3` poly-A tail that help with stability and translation initiation .
- tRNA: These are small RNA molecules that deliver the appropriate amino acid to the ribosome according to the codon on mRNA. Each tRNA has an anticodon loop that is complementary to a specific codon on mRNA, and an acceptor stem that carries a specific amino acid. There are about 20 different types of tRNAs, each corresponding to one of the 20 standard amino acids .
- Aminoacyl tRNA synthetases: These are enzymes that attach the correct amino acid to the corresponding tRNA. There are also about 20 different types of aminoacyl tRNA synthetases, each recognizing one amino acid and one or more tRNAs. These enzymes ensure the accuracy and fidelity of protein synthesis by charging the tRNAs with their proper amino acids .
- Initiation factors: These are proteins that help with the assembly of the ribosome-mRNA complex and the recruitment of the initiator tRNA. In prokaryotes, there are three main initiation factors: IF1, IF2, and IF3. In eukaryotes, there are more than 10 initiation factors involved in translation initiation .
- Elongation factors: These are proteins that facilitate the elongation of the polypeptide chain by bringing in new aminoacyl-tRNAs, translocating the ribosome along the mRNA, and releasing the uncharged tRNAs from the ribosome. In prokaryotes, there are three main elongation factors: EF-Tu, EF-Ts, and EF-G. In eukaryotes, there are similar elongation factors with different names: eEF1A, eEF1B, and eEF2 .
- Termination factors: These are proteins that recognize the stop codons on mRNA and trigger the release of the completed polypeptide chain from the ribosome. In prokaryotes, there are two main termination factors: RF1 and RF2, which bind to different stop codons, and RF3, which helps with their recycling. In eukaryotes, there is only one termination factor: eRF1, which can recognize all three stop codons .
These components work together in a coordinated manner to synthesize proteins according to the genetic code in mRNA. The process of protein synthesis can be divided into three main stages: initiation, elongation, and termination .
Protein synthesis is the process of making proteins from the genetic information encoded in DNA. Proteins are essential for the structure and function of all living cells and organisms. Protein synthesis involves two main stages: transcription and translation.
Transcription is the first stage of protein synthesis. It takes place in the nucleus of eukaryotic cells or in the cytoplasm of prokaryotic cells. Transcription is the process of copying a segment of DNA, called a gene, into a complementary strand of RNA, called messenger RNA (mRNA). The enzyme that catalyzes transcription is called RNA polymerase. RNA polymerase binds to a specific sequence of DNA, called the promoter, that marks the beginning of a gene. RNA polymerase then unwinds the DNA and adds RNA nucleotides that are complementary to the template strand of DNA. For example, if the DNA template strand has the sequence A-T-C-G, then the RNA strand will have the sequence U-A-G-C (U stands for uracil, which replaces thymine in RNA). RNA polymerase continues to add RNA nucleotides until it reaches a termination signal, which marks the end of a gene. The mRNA strand then detaches from the DNA and leaves the nucleus through a nuclear pore.
Translation is the second stage of protein synthesis. It takes place in the cytoplasm of eukaryotic cells or on the plasma membrane of prokaryotic cells. Translation is the process of using the mRNA sequence to assemble amino acids into a polypeptide chain, which is the primary structure of a protein. The molecular machines that carry out translation are called ribosomes. Ribosomes are composed of two subunits: a large subunit and a small subunit. Each subunit is made up of ribosomal RNA (rRNA) and proteins. Ribosomes have three sites for binding molecules: the A site, the P site, and the E site.
Translation involves three main steps: initiation, elongation, and termination.
Initiation is the first step of translation. It involves the assembly of the ribosome on the mRNA and the recruitment of the first amino acid-bearing molecule, called transfer RNA (tRNA). tRNA is a small RNA molecule that has two important features: an anticodon and an amino acid attachment site. The anticodon is a sequence of three nucleotides that is complementary to a codon on mRNA. A codon is a sequence of three nucleotides on mRNA that specifies an amino acid or a stop signal. For example, if the mRNA has the codon A-U-G, then the tRNA with the anticodon U-A-C will bind to it. The amino acid attachment site is where a specific amino acid is covalently linked to tRNA by an enzyme called aminoacyl-tRNA synthetase. There are 20 different amino acids and 20 different aminoacyl-tRNA synthetases that match each amino acid with its corresponding tRNA.
The initiation process differs slightly between prokaryotes and eukaryotes. In prokaryotes, initiation begins when a special tRNA carrying formylmethionine (fMet), which is modified from methionine, binds to the start codon (AUG) on mRNA. This tRNA occupies the P site on the small ribosomal subunit. Then, a protein called initiation factor 3 (IF3) helps to recruit the large ribosomal subunit and form a complete ribosome. In eukaryotes, initiation begins when a special tRNA carrying methionine binds to the start codon (AUG) on mRNA. This tRNA occupies the P site on the small ribosomal subunit. Then, several initiation factors help to scan for the start codon and recruit the large ribosomal subunit and form a complete ribosome.
Elongation is the second step of translation. It involves the sequential addition of amino acids to the growing polypeptide chain. Elongation requires the participation of several elongation factors that facilitate the movement of tRNAs and ribosomes along the mRNA.
Elongation consists of three substeps: codon recognition, peptide bond formation, and translocation.
- Codon recognition: A tRNA carrying an amino acid enters the A site on the ribosome by base-pairing with the next codon on mRNA. The accuracy of this step is ensured by the complementary match between the anticodon and the codon.
- Peptide bond formation: A peptide bond is formed between the amino acid on the tRNA in the A site and the growing polypeptide chain on the tRNA in the P site. This reaction is catalyzed by the rRNA in the large ribosomal subunit, which acts as a ribozyme (an RNA molecule with enzymatic activity). The tRNA in the P site now becomes uncharged, as it has transferred its amino acid to the polypeptide chain.
- Translocation: The ribosome moves one codon along the mRNA in the 5` to 3` direction, shifting the tRNAs from the A site to the P site and from the P site to the E site. The tRNA in the E site is then released from the ribosome and recycled for another amino acid attachment. The A site is now vacant and ready for another tRNA entry.
These substeps are repeated until a stop codon is reached on mRNA.
Termination is the final step of translation. It occurs when the ribosome encounters a stop codon (UAA, UAG, or UGA) on mRNA. Unlike other codons, stop codons do not code for any amino acids. Instead, they are recognized by protein factors called release factors (RFs), which bind to the A site on the ribosome and trigger the release of the polypeptide chain from the tRNA in the P site. The ribosome then dissociates into its two subunits and detaches from the mRNA.
The newly synthesized polypeptide may undergo further modifications, such as folding, cleavage, or addition of other molecules, before becoming a functional protein. Some proteins may also form complexes with other proteins to perform their biological roles.
Protein synthesis is the process of making proteins from the genetic instructions encoded in DNA. It involves two main stages: transcription and translation. Transcription is the copying of a DNA sequence into a messenger RNA (mRNA) molecule in the nucleus. Translation is the decoding of the mRNA sequence into a chain of amino acids (a polypeptide) at a ribosome in the cytoplasm or on the rough endoplasmic reticulum (RER). Each stage can be further divided into several steps.
Translation initiation is the first step of protein synthesis, in which the ribosome, mRNA, and initiator transfer RNA (tRNA) assemble together. The initiator tRNA carries the amino acid methionine (Met) or N-formylmethionine (fMet) in prokaryotes, which corresponds to the start codon AUG on the mRNA. The start codon marks the beginning of the protein-coding sequence (the open reading frame) on the mRNA.
The initiation process differs slightly between prokaryotes and eukaryotes. In prokaryotes, the small ribosomal subunit (30S) binds to a specific sequence on the mRNA called the Shine-Dalgarno sequence, which is located upstream of the start codon. The small subunit then scans the mRNA until it finds the start codon and aligns it with the P site of the ribosome. The initiator fMet-tRNA then binds to the start codon with the help of an initiation factor called IF2, which hydrolyzes guanosine triphosphate (GTP) to provide energy. The large ribosomal subunit (50S) then joins the complex, forming a complete ribosome (70S). The initiation factors IF1 and IF3 are released from the ribosome.
In eukaryotes, the small ribosomal subunit (40S) binds to a modified guanine nucleotide called a cap at the 5` end of the mRNA. The cap helps to protect and stabilize the mRNA. The small subunit then scans the mRNA until it finds the start codon with the help of several initiation factors called eIFs. The initiator Met-tRNA then binds to the start codon with the help of eIF2, which also hydrolyzes GTP to provide energy. The large ribosomal subunit (60S) then joins the complex, forming a complete ribosome (80S). The eIFs are released from the ribosome.
Translation elongation is the second step of protein synthesis, in which amino acids are added one by one to the growing polypeptide chain. Each amino acid is carried by a specific tRNA that has an anticodon complementary to a codon on the mRNA. The elongation process also differs slightly between prokaryotes and eukaryotes.
In both prokaryotes and eukaryotes, elongation involves three main steps: codon recognition, peptide bond formation, and translocation. Codon recognition is when an aminoacyl-tRNA binds to a codon on the A site of the ribosome with the help of an elongation factor called EF-Tu in prokaryotes or eEF1A in eukaryotes, which also hydrolyzes GTP to provide energy. Peptide bond formation is when a peptidyl transferase enzyme catalyzes the formation of a covalent bond between the amino acid on the A site and the growing polypeptide chain on the P site, transferring the chain from the peptidyl-tRNA to the aminoacyl-tRNA. Translocation is when the ribosome moves one codon along the mRNA with the help of another elongation factor called EF-G in prokaryotes or eEF2 in eukaryotes, which also hydrolyzes GTP to provide energy. This movement shifts the uncharged tRNA from the P site to the E site, where it is released, the peptidyl-tRNA from the A site to the P site, where it is ready for another peptide bond formation, and exposes a new codon on the A site for another codon recognition.
The elongation cycle repeats until the ribosome reaches a stop codon on the mRNA.
Translation termination is the final step of protein synthesis, in which the ribosome releases the completed polypeptide chain and the mRNA. Termination is triggered by the encounter of a stop codon on the A site of the ribosome. The stop codons are UAA, UAG, and UGA, and they do not code for any amino acid. Instead, they are recognized by protein factors called release factors (RFs), which bind to the A site and cause the peptidyl transferase to cleave the polypeptide chain from the peptidyl-tRNA on the P site. The RFs also hydrolyze GTP to provide energy for the dissociation of the ribosome into its subunits and the release of the mRNA and the tRNA.
The termination process is similar in prokaryotes and eukaryotes, except for the number and specificity of the RFs. In prokaryotes, there are two RFs: RF1 and RF2. RF1 recognizes the stop codons UAA and UAG, while RF2 recognizes UAA and UGA. A third factor, RF3, assists in the release of RF1 and RF2 from the ribosome. In eukaryotes, there is only one RF: eRF1. eRF1 recognizes all three stop codons. A second factor, eRF3, assists in the binding of eRF1 to the ribosome and the release of the polypeptide chain.
After translation termination, the polypeptide chain may undergo further processing or modification before becoming a functional protein. For example, it may fold into a specific three-dimensional shape, form bonds with other polypeptides or molecules, or be transported to a specific location in the cell or outside the cell.
Protein synthesis is the process of translating the genetic information in mRNA into a sequence of amino acids that forms a polypeptide chain. Protein synthesis is essential for all living organisms, but there are some differences between how it occurs in eukaryotes and prokaryotes. Here are some of the main differences:
- Location: In eukaryotes, protein synthesis occurs in the cytoplasm, where the ribosomes are located. In prokaryotes, protein synthesis occurs in the cytosol, where the DNA, mRNA and ribosomes are all present. Prokaryotes can start protein synthesis even before the transcription of mRNA is completed, which is called coupled transcription-translation .
- Initiation: In eukaryotes, protein synthesis is initiated by the recognition of a start codon (AUG) on the mRNA by a special initiator tRNA that carries methionine. The initiator tRNA binds to the small ribosomal subunit (40S) and then scans the mRNA until it finds the start codon. The large ribosomal subunit (60S) then joins to form the complete ribosome (80S). In prokaryotes, protein synthesis is initiated by the recognition of a specific sequence on the mRNA called the Shine-Dalgarno sequence. This sequence is complementary to a region on the 16S rRNA of the small ribosomal subunit (30S), which allows it to bind to the mRNA near the start codon. The initiator tRNA that carries N-formylmethionine then binds to the start codon (AUG or GUG or UUG). The large ribosomal subunit (50S) then joins to form the complete ribosome (70S).
- Elongation: In eukaryotes and prokaryotes, protein synthesis is elongated by the addition of amino acids to the growing polypeptide chain. This process involves three steps: 1) a charged tRNA enters the A site of the ribosome, where it matches its anticodon with the codon on the mRNA; 2) a peptide bond is formed between the amino acid on the A site and the polypeptide on the P site, catalyzed by peptidyl transferase; 3) the ribosome moves one codon along the mRNA, shifting the tRNAs from A site to P site and from P site to E site, where they are released. This process requires energy from GTP and is facilitated by elongation factors.
- Termination: In eukaryotes and prokaryotes, protein synthesis is terminated when a stop codon (UAA, UAG or UGA) is encountered on the mRNA. The stop codons are recognized by release factors, which bind to the A site and trigger the hydrolysis of the polypeptide from the tRNA on the P site. The ribosome then dissociates into its subunits and releases the mRNA.
Modifications: In eukaryotes, protein synthesis is often followed by post-translational modifications, which are chemical changes that alter or enhance the function of proteins. Some examples of post-translational modifications are phosphorylation, glycosylation, acetylation and ubiquitination. In prokaryotes, post-translational modifications are less common, but some examples are methylation, formylation and lipidation.
Protein Synthesis Inhibitors
Protein synthesis inhibitors are substances that stop or slow the growth or proliferation of cells by disrupting the processes that lead directly to the generation of new proteins. They usually act at the ribosome level, taking advantage of the major differences between prokaryotic and eukaryotic ribosome structures. Protein synthesis inhibitors are often used as antimicrobial agents to treat bacterial infections, as well as some fungal and protozoal diseases.
There are different types of protein synthesis inhibitors that target different stages of the translation process, such as initiation, elongation and termination. The following table summarizes some of the common protein synthesis inhibitors and their mechanisms of action.
|Rifamycin||Inhibits bacterial DNA transcription into mRNA by binding to DNA-dependent RNA polymerase.||Earlier stage|
|α-Amanitin||Inhibits eukaryotic DNA transcription machinery.||Earlier stage|
|Linezolid||Prevents the formation of the initiation complex by binding to the 23S rRNA of the 50S ribosomal subunit.||Initiation|
|Aminoglycosides||Prevent ribosome assembly by binding to the 30S ribosomal subunit. Interfere with the proofreading process, causing increased rate of error and premature termination. Inhibit ribosomal translocation.||Initiation, elongation and translocation|
|Tetracyclines||Block the A site on the ribosome, preventing the binding of aminoacyl-tRNAs.||Elongation|
|Chloramphenicol||Blocks the peptidyl transfer step of elongation on the 50S ribosomal subunit in both bacteria and mitochondria.||Elongation|
|Macrolides||Inhibit peptidyl transfer and ribosomal translocation by binding to the 23S rRNA of the 50S ribosomal subunit.||Elongation and translocation|
|Quinupristin/dalfopristin||Act synergistically to inhibit peptidyl transfer and ribosomal translocation by binding to different sites on the 50S ribosomal subunit.||Elongation and translocation|
|Geneticin (G418)||Inhibits elongation in both prokaryotic and eukaryotic ribosomes.||Elongation|
|Trichothecene mycotoxins||Potent and non-selective inhibitors of peptide elongation.||Elongation|
|Puromycin||Mimics aminoacyl-tRNA and releases incomplete polypeptide chains from the ribosome.||Elongation|
|Streptomycin||Affects the binding activity of formyl methionyl-tRNA to ribosomes, preventing correct initiation.||Initiation|
|Release factors (RF1, RF2, RF3)||Recognize stop codons and catalyze the release of polypeptide chains from the ribosome.||Termination|
Protein synthesis inhibitors can have various effects on cells, such as reducing growth rate, inducing stress responses, altering gene expression, triggering apoptosis or autophagy, or modulating immune responses. Some protein synthesis inhibitors can also have beneficial effects on human health, such as anti-inflammatory, anti-cancer, neuroprotective or anti-aging properties.
Protein synthesis inhibitors are important tools for studying cellular functions and mechanisms, as well as potential therapeutic agents for various diseases. However, they also pose some challenges, such as toxicity, resistance, specificity and delivery. Therefore, further research is needed to optimize their use and discover new compounds with improved properties.
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