Protein Synthesis in Eukaryotes- Definition, Enzymes and Process

Ribosomes are the molecular machines responsible for protein synthesis, or translation, in the cell. They are made of ribosomal RNA (rRNA) and proteins, and consist of two subunits: a large subunit and a small subunit. The large subunit sits on top of the small subunit, with a messenger RNA (mRNA) template sandwiched between them.

Protein synthesis is the process by which the genetic information encoded in mRNA is translated into a specific sequence of amino acids, which are the building blocks of proteins. Proteins perform various functions in the cell, such as catalyzing biochemical reactions, forming structures, transporting molecules, and regulating gene expression.

The ribosomes receive their orders for protein synthesis from the nucleus, where portions of DNA (genes) are transcribed to make mRNA. The mRNA travels from the nucleus to the cytoplasm, where it binds to a ribosome near its 5` end. The ribosome then reads the mRNA sequence from the 5` to 3` direction, using transfer RNA (tRNA) molecules to bring the corresponding amino acids to the growing polypeptide chain.

The ribosome has three binding sites for tRNA: the aminoacyl-tRNA site (A site), where the incoming tRNA with its attached amino acid binds; the peptidyl-tRNA site (P site), where the tRNA linked to the growing polypeptide chain is bound; and the exit site (E site), where the tRNA leaves the ribosome after its role in translation. The ribosome catalyzes the formation of a peptide bond between the amino acids at the A and P sites, and then shifts the mRNA by one codon, moving the tRNAs from one site to another. This cycle repeats until the ribosome reaches a stop codon on the mRNA, signaling the end of translation.

Ribosomes can be found either freely floating in the cytosol or attached to the endoplasmic reticulum (ER), a membranous organelle that synthesizes lipids and modifies proteins. The location of ribosomes determines where the proteins they produce end up: proteins synthesized by free ribosomes usually stay in the cytosol or are transported to other organelles, while proteins synthesized by ER-bound ribosomes are usually destined for secretion or insertion into membranes.

Ribosomes are essential for life, as they enable cells to produce proteins that carry out various functions. In eukaryotes, ribosomes are larger and more complex than in prokaryotes, reflecting their different evolutionary origins and cellular needs. In this article, we will explore how protein synthesis occurs in eukaryotes, and how it differs from protein synthesis in prokaryotes.

Ribosomes are the molecular machines that synthesize proteins from mRNA templates. They are composed of two subunits, each containing ribosomal RNA (rRNA) and proteins. The size and composition of ribosomes differ between eukaryotes and prokaryotes, as summarized in the table below:

The sedimentation coefficient is a measure of how fast a particle settles in a centrifugal field. It is expressed in Svedberg units (S), which are proportional to the molecular weight and shape of the particle. Eukaryotic ribosomes have a higher sedimentation coefficient than prokaryotic ribosomes because they are larger and more complex.

The subunit sizes are also expressed in Svedberg units, but they are not additive. For example, the 80S eukaryotic ribosome is not composed of a 40S and a 40S subunit, but rather a 40S and a 60S subunit. The discrepancy is due to the shape and interaction of the subunits, which affect their sedimentation behavior.

The rRNA types refer to the different rRNA molecules that make up the ribosomal subunits. Eukaryotic ribosomes have four types of rRNA: 18S in the small subunit, and 5.8S, 28S and 5S in the large subunit. Prokaryotic ribosomes have three types of rRNA: 16S in the small subunit, and 23S and 5S in the large subunit. The numbers indicate the approximate length of the rRNA molecules in Svedberg units.

The rRNA length is the total number of nucleotides in the rRNA molecules of each ribosome. Eukaryotic ribosomes have longer rRNA than prokaryotic ribosomes, reflecting their higher complexity and diversity.

The protein number is the total number of proteins in each ribosome. Eukaryotic ribosomes have more proteins than prokaryotic ribosomes, again reflecting their higher complexity and diversity.

The protein mass is the total mass of proteins in each ribosome. Eukaryotic ribosomes have heavier proteins than prokaryotic ribosomes, due to their larger size and higher number.

The differences between eukaryotic and prokaryotic ribosomes have implications for their function and regulation. For example, eukaryotic ribosomes are more sensitive to certain antibiotics that target prokaryotic ribosomes, such as erythromycin and chloramphenicol. Moreover, eukaryotic ribosomes have more initiation factors and elongation factors than prokaryotic ribosomes, reflecting their more elaborate mechanisms of translation initiation and elongation.

Each eukaryotic ribosome has three binding sites for tRNAs: the aminoacyl-tRNA binding site (or A site), the peptidyl-tRNA binding site (or P site), and the exit site (or E site). These sites are formed by the rRNA molecules in the ribosome and play important roles in the elongation stage of protein synthesis.

The A site is where the incoming aminoacyl-tRNA binds to the codon on the mRNA. The aminoacyl-tRNA is a tRNA molecule that carries a specific amino acid at its 3` end. The anticodon of the aminoacyl-tRNA is complementary to the codon of the mRNA. The correct pairing of the codon and anticodon ensures that the amino acid sequence of the protein matches the genetic information in the mRNA.

The P site is where the tRNA linked to the growing polypeptide chain is bound. The polypeptide chain is attached to the 3` end of the tRNA by a peptide bond. The peptide bond is formed between the amino group of the amino acid in the A site and the carboxyl group of the amino acid in the P site. The formation of the peptide bond is catalyzed by a ribozyme, which is an RNA molecule that acts as an enzyme.

The E site is a binding site for tRNA following its role in translation and prior to its release from the ribosome. The tRNA in the E site is deacylated, meaning that it has no amino acid attached to it. The deacylated tRNA leaves the ribosome and can be recharged with a new amino acid by an aminoacyl-tRNA synthetase.

During elongation, the ribosome moves along the mRNA from 5` to 3` direction, synthesizing the protein from N-terminal to C-terminal direction. As the ribosome moves, it shifts the tRNAs from one site to another. The tRNA that was in the P site moves to the E site and exits the ribosome. The tRNA that was in the A site moves to the P site and transfers its amino acid to the polypeptide chain. A new aminoacyl-tRNA enters the A site and binds to the next codon on the mRNA. This cycle repeats until a stop codon is reached on the mRNA, which signals the termination of protein synthesis.

The A, P, and E sites are essential for ensuring that protein synthesis proceeds accurately and efficiently. They allow for precise recognition of codons and anticodons, formation of peptide bonds, and translocation of tRNAs and mRNA. By using these sites, eukaryotic ribosomes can synthesize proteins with complex structures and functions.

Protein synthesis (or translation) is the process of decoding the genetic information in mRNA and building the corresponding polypeptide chain. In eukaryotes, protein synthesis takes place in three main stages: initiation, elongation, and termination. Each stage involves specific ribosomal subunits, tRNAs, and protein factors that facilitate the accurate and efficient translation of mRNA.

Initiation

Initiation is the stage where the ribosome assembles on the mRNA and finds the start codon (usually AUG) that signals the beginning of protein synthesis. In eukaryotes, initiation requires at least nine distinct eukaryotic initiation factors (eIFs) that help to form a pre-initiation complex consisting of the 40S small ribosomal subunit, Met-tRNAimet (the initiator tRNA charged with methionine), eIF-2, and GTP. The pre-initiation complex binds to the 5` end of the mRNA, which is marked by a cap structure (7-methylguanosine) that is recognized by eIF-4F (also called cap-binding complex). The complex then scans along the mRNA in a 5` to 3` direction until it encounters the initiation codon, which is often (but not always) contained in a short sequence called the Kozak consensus (5`-ACCAUGG-3`). Once the initiation codon is recognized by the anticodon of Met-tRNAimet, the 60S large ribosomal subunit joins to form an 80S initiation complex, which is ready to start elongation. The joining of the 60S subunit requires the hydrolysis of GTP and leads to the release of several initiation factors.

Elongation

Elongation is the stage where the ribosome moves along the mRNA and adds amino acids to the growing polypeptide chain. In eukaryotes, elongation depends on three elongation factors: eEF-1A, eEF-1B, and eEF-2, which have similar functions to their prokaryotic counterparts EF-Tu, EF-Ts and EF-G. Elongation consists of three steps that are repeated for each codon in the mRNA:

• Positioning: The correct aminoacyl-tRNA (a tRNA charged with an amino acid) is delivered to the A site (aminoacyl-tRNA binding site) of the ribosome by eEF-1A and GTP. The anticodon of the aminoacyl-tRNA must match the codon in the mRNA for accurate translation.
• Peptide bond formation: The amino acid in the A site is transferred to the growing polypeptide chain in the P site (peptidyl-tRNA binding site) by a catalytic reaction mediated by the rRNA in the large ribosomal subunit. This reaction forms a peptide bond between the two amino acids and releases the tRNA in the P site.
• Translocation: The ribosome moves one codon forward along the mRNA by eEF-2 and GTP. This shifts the tRNA in the A site to the P site and leaves the A site vacant for the next aminoacyl-tRNA. The deacylated tRNA in the P site is ejected from the ribosome.

These steps are repeated until a stop codon (UAA, UAG, or UGA) is reached in the mRNA.

Termination

Termination is the stage where protein synthesis stops and the ribosome disassembles from the mRNA. In eukaryotes, termination requires two release factors: eRF-1 and eRF-3. When a stop codon enters the A site of the ribosome, eRF-1 recognizes it and binds to it with the help of eRF-3 and GTP. This triggers a hydrolysis reaction that releases the completed polypeptide from the tRNA in the P site. The ribosome then dissociates into its subunits and releases the mRNA.

The newly synthesized polypeptide may undergo further modifications such as folding, cleavage, or addition of other molecules before becoming a functional protein.

Although the basic mechanism of protein synthesis is similar in both prokaryotes and eukaryotes, there are some notable differences that reflect the evolutionary divergence and complexity of the two domains. Some of the main differences are:

• Ribosome size and composition: Prokaryotic ribosomes have a sedimentation coefficient of 70S and are composed of two subunits: 30S and 50S. Eukaryotic ribosomes have a sedimentation coefficient of 80S and are composed of two subunits: 40S and 60S. The subunits of eukaryotic ribosomes are also more complex than those of prokaryotic ribosomes, containing more proteins and rRNAs.
• mRNA structure and processing: Prokaryotic mRNAs are usually polycistronic, meaning that they encode more than one protein. Each protein-coding sequence has its own start and stop codons. Eukaryotic mRNAs are usually monocistronic, meaning that they encode only one protein. Eukaryotic mRNAs also undergo extensive processing in the nucleus before being exported to the cytoplasm for translation. This includes the addition of a 5` cap, a poly(A) tail, and splicing to remove introns.
• Initiation factors and codons: Initiation of protein synthesis in prokaryotes requires three initiation factors (IFs): IF-1, IF-2, and IF-3. Initiation of protein synthesis in eukaryotes requires at least nine initiation factors (eIFs): eIF-1, eIF-1A, eIF-2, eIF-3, eIF-4A, eIF-4E, eIF-4G, eIF-5, and eIF-6. The initiating amino acid in prokaryotes is N-formylmethionine (fMet), whereas in eukaryotes it is methionine (Met). The initiation codon in prokaryotes is usually AUG, but sometimes GUG or UUG can also be used. The initiation codon in eukaryotes is almost always AUG. In prokaryotes, the initiation codon is recognized by a complementary sequence on the 16S rRNA of the 30S subunit called the Shine-Dalgarno sequence. In eukaryotes, the initiation codon is recognized by scanning the mRNA from the 5` end until a Kozak consensus sequence is found.
• Elongation factors and codons: Elongation of protein synthesis in prokaryotes requires three elongation factors (EFs): EF-Tu, EF-Ts, and EF-G. Elongation of protein synthesis in eukaryotes requires three elongation factors (eEFs): eEF-1A, eEF-1B, and eEF-2. The functions of these factors are similar in both domains, but their structures are different. Some codons have different meanings in prokaryotes and eukaryotes. For example, UGA is a stop codon in most prokaryotes, but it encodes selenocysteine (Sec) in some bacteria and archaea. UGA also encodes Sec in some eukaryotes, but only when a specific RNA structure called SECIS element is present downstream of the codon. Similarly, UAG is a stop codon in most prokaryotes, but it encodes pyrrolysine (Pyl) in some archaea and bacteria. UAG also encodes Pyl in some eukaryotes, but only when a specific tRNA is present.
• Termination factors and codons: Termination of protein synthesis in prokaryotes requires two release factors (RFs): RF-1 and RF-2. RF-1 recognizes UAA and UAG as stop codons, while RF-2 recognizes UAA and UGA as stop codons. Termination of protein synthesis in eukaryotes requires only one release factor (eRF): eRF-1. eRF-1 recognizes all three stop codons: UAA, UAG, and UGA. Both prokaryotic and eukaryotic release factors require a third factor called RF-3 or eRF-3 respectively to facilitate their binding and release from the ribosome.

These differences between protein synthesis in prokaryotes and eukaryotes reflect the adaptations that each domain has evolved to optimize their gene expression and cellular function.

The first step of protein synthesis in eukaryotes is the formation of a pre-initiation complex consisting of the following components:

• The 40S small ribosomal subunit, which contains the 18S rRNA and several proteins.
• The initiator tRNA, which is charged with the amino acid methionine and has the anticodon 3`-UAC-5` that matches the start codon 5`-AUG-3` on the mRNA. This tRNA is also called Met-tRNAimet to distinguish it from the tRNA that carries methionine at internal positions of the polypeptide chain.
• The eukaryotic initiation factor 2 (eIF2), which is a heterotrimeric protein that binds to Met-tRNAimet and GTP. eIF2 facilitates the delivery of Met-tRNAimet to the small ribosomal subunit.
• The eukaryotic initiation factor 3 (eIF3), which is a large multisubunit protein that binds to the small ribosomal subunit and prevents its premature association with the large ribosomal subunit. eIF3 also helps to recruit other initiation factors and mRNA to the small ribosomal subunit.

The pre-initiation complex binds to the 5` end of the eukaryotic mRNA, a step that requires another initiation factor called eukaryotic initiation factor 4F (eIF4F). eIF4F is also known as the cap-binding complex because it recognizes and binds to the 7-methylguanosine cap that is present at the 5` end of most eukaryotic mRNAs. eIF4F consists of three subunits:

• The eukaryotic initiation factor 4E (eIF4E), which directly binds to the cap structure and acts as a scaffold for the other subunits.
• The eukaryotic initiation factor 4G (eIF4G), which interacts with eIF4E, eIF3, and another factor called poly(A)-binding protein (PABP). PABP binds to the poly(A) tail that is present at the 3` end of most eukaryotic mRNAs, forming a circular structure that enhances translation efficiency and stability.
• The eukaryotic initiation factor 4A (eIF4A), which is an ATP-dependent RNA helicase that unwinds any secondary structures in the mRNA, preparing it for translation.

The complex then moves along the mRNA in a 5` to 3` direction until it locates the AUG initiation codon. This process is called scanning and requires ATP hydrolysis by eIF4A. The initiation codon is usually recognizable because it is often (but not always) contained in a short sequence called the Kozak consensus (5`-ACCAUGG-3`), which enhances its recognition by the initiator tRNA.

Once the complex is positioned over the initiation codon, the 60S large ribosomal subunit binds to form an 80S initiation complex, a step that requires the hydrolysis of GTP by eIF2 and leads to the release of several initiation factors. The initiator tRNA occupies the P site in the ribosome, and the A site is ready to receive an aminoacyl-tRNA. The initiation phase of protein synthesis in eukaryotes is now complete and elongation can begin.

Elongation is the process of adding amino acids to the growing polypeptide chain by forming peptide bonds between them. Elongation depends on eukaryotic elongation factors, which are proteins that facilitate the movement and interaction of the ribosome, mRNA and tRNAs.

There are three main steps in the elongation cycle:

1. Positioning the correct aminoacyl-tRNA in the A site of the ribosome. This step requires the elongation factor eEF-1A, which binds to an aminoacyl-tRNA and delivers it to the A site, where it can base-pair with the codon on the mRNA. The eEF-1A also hydrolyzes GTP to GDP and releases it along with the aminoacyl-tRNA. The accuracy of this step is ensured by a proofreading mechanism that rejects incorrect aminoacyl-tRNAs from the A site.
2. Forming the peptide bond between the amino acids in the P and A sites. This step is catalyzed by the peptidyl transferase activity of the large ribosomal subunit, which transfers the growing polypeptide chain from the tRNA in the P site to the amino acid in the A site. This results in a longer polypeptide attached to the tRNA in the A site, and a deacylated tRNA in the P site.
3. Shifting the mRNA by one codon relative to the ribosome. This step requires the elongation factor eEF-2, which binds to the ribosome and promotes its translocation along the mRNA. The eEF-2 also hydrolyzes GTP to GDP and releases it along with the ribosome. This movement causes the tRNA in the A site to move to the P site, and the tRNA in the P site to move to the E site, where it can exit the ribosome. The A site is now vacant and ready to receive a new aminoacyl-tRNA.

These steps are repeated until a stop codon is encountered on the mRNA, which signals the termination of protein synthesis.

The rate of elongation in eukaryotes is slower than in prokaryotes, due to the larger size and complexity of eukaryotic ribosomes and mRNA. However, eukaryotes can compensate for this by initiating multiple rounds of translation on a single mRNA, forming a structure called a polysome or polyribosome.

The final stage of protein synthesis is termination, which occurs when the ribosome reaches a stop codon on the mRNA. There are three stop codons in the genetic code: UAA, UAG, and UGA. These codons do not encode any amino acid, but signal the end of translation.

In eukaryotes, termination of protein synthesis requires two main factors: eukaryotic release factor 1 (eRF1) and eukaryotic release factor 3 (eRF3). eRF1 is a protein that recognizes all three stop codons and binds to the A site of the ribosome. eRF3 is a GTPase that interacts with eRF1 and facilitates its binding to the ribosome.

The binding of eRF1 to the stop codon triggers a series of events that lead to the release of the completed polypeptide chain from the ribosome. First, eRF1 catalyzes the hydrolysis of the ester bond between the last amino acid and its tRNA in the P site. This frees the polypeptide chain from the tRNA and transfers it to eRF1. Second, eRF3 hydrolyzes its bound GTP and causes a conformational change in eRF1 that releases the polypeptide chain from the A site. Third, eRF1 and eRF3 dissociate from the ribosome, leaving behind a post-termination complex consisting of the mRNA, the deacylated tRNA in the P site, and the two ribosomal subunits.

The post-termination complex is then recycled by another set of factors that prepare the ribosome for a new round of translation. These factors include eukaryotic initiation factor 3 (eIF3), which binds to the 40S subunit and prevents its reassociation with the 60S subunit; eukaryotic initiation factor 1A (eIF1A), which binds to the A site and blocks its occupancy by tRNA; and ABCE1 (ATP-binding cassette subfamily E member 1), which is an ATPase that binds to the 50S subunit and stimulates its dissociation from the mRNA and the tRNA. The released mRNA can then be degraded or reused for another translation cycle.

Termination of protein synthesis is a highly regulated process that ensures the accuracy and efficiency of protein production. It also plays a role in quality control by detecting and eliminating aberrant mRNAs or polypeptides that may arise due to errors in transcription, splicing, or translation. For example, some mRNAs may contain premature stop codons that result in truncated proteins. These mRNAs are recognized and degraded by a mechanism called nonsense-mediated decay (NMD), which involves several factors that interact with eRFs and monitor the position of the stop codon relative to the exon-exon junctions on the mRNA. Similarly, some polypeptides may contain misfolded or damaged regions that impair their function or stability. These polypeptides are targeted and degraded by a mechanism called ubiquitin-proteasome system (UPS), which involves covalent attachment of ubiquitin molecules to the defective polypeptides and their subsequent degradation by proteasomes.

Termination of protein synthesis is thus an essential step in gene expression that ensures the fidelity and quality of protein production in eukaryotic cells.

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