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11.4: Protein Synthesis (Translation) - Biology

11.4: Protein Synthesis (Translation) - Biology


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Learning Objectives

  • Describe the genetic code and explain why it is considered almost universal
  • Explain the process of translation and the functions of the molecular machinery of translation
  • Compare translation in eukaryotes and prokaryotes

The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. They perform virtually every function of a cell, serving as both functional (e.g., enzymes) and structural elements. The process of translation, or protein synthesis, the second part of gene expression, involves the decoding by a ribosome of an mRNA message into a polypeptide product.

The Genetic Code

Translation of the mRNA template converts nucleotide-based genetic information into the “language” of amino acids to create a protein product. A protein sequence consists of 20 commonly occurring amino acids. Each amino acid is defined within the mRNA by a triplet of nucleotides called a codon. The relationship between an mRNA codon and its corresponding amino acid is called the genetic code.

The three-nucleotide code means that there is a total of 64 possible combinations (43, with four different nucleotides possible at each of the three different positions within the codon). This number is greater than the number of amino acids and a given amino acid is encoded by more than one codon (Figure (PageIndex{1})). This redundancy in the genetic code is called degeneracy. Typically, whereas the first two positions in a codon are important for determining which amino acid will be incorporated into a growing polypeptide, the third position, called the wobble position, is less critical. In some cases, if the nucleotide in the third position is changed, the same amino acid is still incorporated.

Whereas 61 of the 64 possible triplets code for amino acids, three of the 64 codons do not code for an amino acid; they terminate protein synthesis, releasing the polypeptide from the translation machinery. These are called stop codons or nonsense codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also typically serves as the start codon to initiate translation. The reading frame, the way nucleotides in mRNA are grouped into codons, for translation is set by the AUG start codon near the 5’ end of the mRNA. Each set of three nucleotides following this start codon is a codon in the mRNA message.

The genetic code is nearly universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all extant life on earth shares a common origin. However, unusual amino acids such as selenocysteine and pyrrolysine have been observed in archaea and bacteria. In the case of selenocysteine, the codon used is UGA (normally a stop codon). However, UGA can encode for selenocysteine using a stem-loop structure (known as the selenocysteine insertion sequence, or SECIS element), which is found at the 3’ untranslated region of the mRNA. Pyrrolysine uses a different stop codon, UAG. The incorporation of pyrrolysine requires the pylS gene and a unique transfer RNA (tRNA) with a CUA anticodon.

Exercise (PageIndex{1})

  1. How many bases are in each codon?
  2. What amino acid is coded for by the codon AAU?
  3. What happens when a stop codon is reached?

The Protein Synthesis Machinery

In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation. The composition of each component varies across taxa; for instance, ribosomes may consist of different numbers of ribosomal RNAs (rRNAs) and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.

Ribosomes

A ribosome is a complex macromolecule composed of catalytic rRNAs (called ribozymes) and structural rRNAs, as well as many distinct polypeptides. Mature rRNAs make up approximately 50% of each ribosome. Prokaryotes have 70S ribosomes, whereas eukaryotes have 80S ribosomes in the cytoplasm and rough endoplasmic reticulum, and 70S ribosomes in mitochondria and chloroplasts. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation. In E. coli, the small subunit is described as 30S (which contains the 16S rRNA subunit), and the large subunit is 50S (which contains the 5S and 23S rRNA subunits), for a total of 70S (Svedberg units are not additive). Eukaryote ribosomes have a small 40S subunit (which contains the 18S rRNA subunit) and a large 60S subunit (which contains the 5S, 5.8S and 28S rRNA subunits), for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit binds tRNAs (discussed in the next subsection).

Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5’ to 3’ and synthesizing the polypeptide from the N terminus to the C terminus. The complete structure containing an mRNA with multiple associated ribosomes is called a polyribosome (or polysome). In both bacteria and archaea, before transcriptional termination occurs, each protein-encoding transcript is already being used to begin synthesis of numerous copies of the encoded polypeptide(s) because the processes of transcription and translation can occur concurrently, forming polyribosomes (Figure (PageIndex{2})). The reason why transcription and translation can occur simultaneously is because both of these processes occur in the same 5’ to 3’ direction, they both occur in the cytoplasm of the cell, and because the RNA transcript is not processed once it is transcribed. This allows a prokaryotic cell to respond to an environmental signal requiring new proteins very quickly. In contrast, in eukaryotic cells, simultaneous transcription and translation is not possible. Although polyribosomes also form in eukaryotes, they cannot do so until RNA synthesis is complete and the RNA molecule has been modified and transported out of the nucleus.

Transfer RNAs

Transfer RNAs (tRNAs) are structural RNA molecules and, depending on the species, many different types of tRNAs exist in the cytoplasm. Bacterial species typically have between 60 and 90 types. Serving as adaptors, each tRNA type binds to a specific codon on the mRNA template and adds the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. The tRNA molecule interacts with three factors: aminoacyl tRNA synthetases, ribosomes, and mRNA.

Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the single-stranded RNA molecule hydrogen bond with each other (Figure (PageIndex{3})). This shape positions the amino-acid binding site, called the CCA amino acid binding end, which is a cytosine-cytosine-adenine sequence at the 3’ end of the tRNA, and the anticodonat the other end. The anticodon is a three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing.

An amino acid is added to the end of a tRNA molecule through the process of tRNA “charging,” during which each tRNA molecule is linked to its correct or cognate amino acid by a group of enzymes called aminoacyl tRNA synthetases. At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids. During this process, the amino acid is first activated by the addition of adenosine monophosphate (AMP) and then transferred to the tRNA, making it a charged tRNA, and AMP is released.

Exercise (PageIndex{2})

  1. Describe the structure and composition of the prokaryotic ribosome.
  2. In what direction is the mRNA template read?
  3. Describe the structure and function of a tRNA.

The Mechanism of Protein Synthesis

Translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between bacterial and eukaryotic translation.

Initiation

The initiation of protein synthesis begins with the formation of an initiation complex. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-methionine(fMet-tRNAfMet) (Figure (PageIndex{4})). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.

In eukaryotes, initiation complex formation is similar, with the following differences:

  • The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi
  • Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5’ cap of the eukaryotic mRNA, then tracks along the mRNA in the 5’ to 3’ direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.

Elongation

In prokaryotes and eukaryotes, the basics of elongation of translation are the same. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites: The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one notable exception to this assembly line of tRNAs: During initiation complex formation, bacterial fMet−tRNAfMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3’ direction. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.

Termination

The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation init iation complex.

In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are illustrated in Figure (PageIndex{5}) and listed in Figure (PageIndex{6}).

Protein Targeting, Folding, and Modification

During and after translation, polypeptides may need to be modified before they are biologically active. Post-translational modifications include:

  1. removal of translated signal sequences—short tails of amino acids that aid in directing a protein to a specific cellular compartment
  2. proper “folding” of the polypeptide and association of multiple polypeptide subunits, often facilitated by chaperone proteins, into a distinct three-dimensional structure
  3. proteolytic processing of an inactive polypeptide to release an active protein component, and
  4. various chemical modifications (e.g., phosphorylation, methylation, or glycosylation) of individual amino acids.
  • What are the components of the initiation complex for translation in prokaryotes?
  • What are two differences between initiation of prokaryotic and eukaryotic translation?
  • What occurs at each of the three active sites of the ribosome?
  • What causes termination of translation?

Key Concepts and Summary

  • In translation, polypeptides are synthesized using mRNA sequences and cellular machinery, including tRNAs that match mRNA codons to specific amino acids and ribosomes composed of RNA and proteins that catalyze the reaction.
  • The genetic code is degenerate in that several mRNA codons code for the same amino acids. The genetic code is almost universal among living organisms.
  • Prokaryotic (70S) and cytoplasmic eukaryotic (80S) ribosomes are each composed of a large subunit and a small subunit of differing sizes between the two groups. Each subunit is composed of rRNA and protein. Organelle ribosomes in eukaryotic cells resemble prokaryotic ribosomes.
  • Some 60 to 90 species of tRNA exist in bacteria. Each tRNA has a three-nucleotide anticodon as well as a binding site for a cognate amino acid. All tRNAs with a specific anticodon will carry the same amino acid.
  • Initiation of translation occurs when the small ribosomal subunit binds with initiation factors and an initiator tRNA at the start codon of an mRNA, followed by the binding to the initiation complex of the large ribosomal subunit.
  • In prokaryotic cells, the start codon codes for N-formyl-methionine carried by a special initiator tRNA. In eukaryotic cells, the start codon codes for methionine carried by a special initiator tRNA. In addition, whereas ribosomal binding of the mRNA in prokaryotes is facilitated by the Shine-Dalgarno sequence within the mRNA, eukaryotic ribosomes bind to the 5’ cap of the mRNA.
  • During the elongation stage of translation, a charged tRNA binds to mRNA in the A site of the ribosome; a peptide bond is catalyzed between the two adjacent amino acids, breaking the bond between the first amino acid and its tRNA; the ribosome moves one codon along the mRNA; and the first tRNA is moved from the P site of the ribosome to the E site and leaves the ribosomal complex.
  • Termination of translation occurs when the ribosome encounters a stop codon, which does not code for a tRNA. Release factors cause the polypeptide to be released, and the ribosomal complex dissociates.
  • In prokaryotes, transcription and translation may be coupled, with translation of an mRNA molecule beginning as soon as transcription allows enough mRNA exposure for the binding of a ribosome, prior to transcription termination. Transcription and translation are not coupled in eukaryotes because transcription occurs in the nucleus, whereas translation occurs in the cytoplasm or in association with the rough endoplasmic reticulum.
  • Polypeptides often require one or more post-translational modifications to become biologically active.

Multiple Choice

Which of the following is the name of the three-base sequence in the mRNA that binds to a tRNA molecule?

A. P site
B. codon
C. anticodon
D. CCA binding site

B

Which component is the last to join the initiation complex during the initiation of translation?

A. the mRNA molecule
B. the small ribosomal subunit
C. the large ribosomal subunit
D. the initiator tRNA

C

During elongation in translation, to which ribosomal site does an incoming charged tRNA molecule bind?

A. A site
B. P site
C. E site
D. B site

A

Which of the following is the amino acid that appears at the N-terminus of all newly translated prokaryotic and eukaryotic polypeptides?

A. tryptophan
B. methionine
C. selenocysteine
D. glycine

B

When the ribosome reaches a nonsense codon, which of the following occurs?

A. a methionine is incorporated
B. the polypeptide is released
C. a peptide bond forms
D. the A site binds to a charged tRNA

B

Fill in the Blank

The third position within a codon, in which changes often result in the incorporation of the same amino acid into the growing polypeptide, is called the ________.

wobble position

The enzyme that adds an amino acid to a tRNA molecule is called ________.

aminoacyl-tRNA synthetase

True/False

Each codon within the genetic code encodes a different amino acid.

False

Short Answer

Why does translation terminate when the ribosome reaches a stop codon? What happens?

How does the process of translation differ between prokaryotes and eukaryotes?

What is meant by the genetic code being nearly universal?

Below is an antisense DNA sequence. Translate the mRNA molecule synthesized using the genetic code, recording the resulting amino acid sequence, indicating the N and C termini.

Antisense DNA strand: 3’-T A C T G A C T G A C G A T C-5’

Critical Thinking

Label the following in the figure: ribosomal E, P, and A sites; mRNA; codons; anticodons; growing polypeptide; incoming amino acid; direction of translocation; small ribosomal unit; large ribosomal unit.

Prior to the elucidation of the genetic code, prominent scientists, including Francis Crick, had predicted that each mRNA codon, coding for one of the 20 amino acids, needed to be at least three nucleotides long. Why is it not possible for codons to be any shorter?


9.4 Translation

The synthesis of proteins is one of a cell’s most energy-consuming metabolic processes. In turn, proteins account for more mass than any other component of living organisms (with the exception of water), and proteins perform a wide variety of the functions of a cell. The process of translation, or protein synthesis, involves decoding an mRNA message into a polypeptide product. Amino acids are covalently strung together in lengths ranging from approximately 50 amino acids to more than 1,000.

The Protein Synthesis Machinery

In addition to the mRNA template, many other molecules contribute to the process of translation. The composition of each component may vary across species for instance, ribosomes may consist of different numbers of ribosomal RNAs ( rRNA ) and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors (Figure 9.19).

In E. coli, there are 200,000 ribosomes present in every cell at any given time. A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs.

Ribosomes are located in the cytoplasm in prokaryotes and in the cytoplasm and endoplasmic reticulum of eukaryotes. Ribosomes are made up of a large and a small subunit that come together for translation. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs , a type of RNA molecule that brings amino acids to the growing chain of the polypeptide. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction.

Depending on the species, 40 to 60 types of tRNA exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. For each tRNA to function, it must have its specific amino acid bonded to it. In the process of tRNA “charging,” each tRNA molecule is bonded to its correct amino acid.

The Genetic Code

To summarize what we know to this point, the cellular process of transcription generates messenger RNA (mRNA), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 commonly occurring amino acids therefore, it can be said that the protein alphabet consists of 20 letters. Each amino acid is defined by a three-nucleotide sequence called the triplet codon . The relationship between a nucleotide codon and its corresponding amino acid is called the genetic code .

Given the different numbers of “letters” in the mRNA and protein “alphabets,” combinations of nucleotides corresponded to single amino acids. Using a three-nucleotide code means that there are a total of 64 (4 × 4 × 4) possible combinations therefore, a given amino acid is encoded by more than one nucleotide triplet (Figure 9.20).

Three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called stop codons . Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5' end of the mRNA. The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all life on Earth shares a common origin.

The Mechanism of Protein Synthesis

Just as with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.

Protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small ribosome subunit, the mRNA template, three initiation factors, and a special initiator tRNA. The initiator tRNA interacts with the AUG start codon, and links to a special form of the amino acid methionine that is typically removed from the polypeptide after translation is complete.

In prokaryotes and eukaryotes, the basics of polypeptide elongation are the same, so we will review elongation from the perspective of E. coli. The large ribosomal subunit of E. coli consists of three compartments: the A site binds incoming charged tRNAs (tRNAs with their attached specific amino acids). The P site binds charged tRNAs carrying amino acids that have formed bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E site releases dissociated tRNAs so they can be recharged with free amino acids. The ribosome shifts one codon at a time, catalyzing each process that occurs in the three sites. With each step, a charged tRNA enters the complex, the polypeptide becomes one amino acid longer, and an uncharged tRNA departs. The energy for each bond between amino acids is derived from GTP, a molecule similar to ATP (Figure 9.21). Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid polypeptide could be translated in just 10 seconds.

Termination of translation occurs when a stop codon (UAA, UAG, or UGA) is encountered. When the ribosome encounters the stop codon, the growing polypeptide is released and the ribosome subunits dissociate and leave the mRNA. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

Concepts in Action

Transcribe a gene and translate it to protein using complementary pairing and the genetic code at this site.


  1. What are the roles of mRNA and tRNA in protein synthesis?
  2. What is the initiation codon?
  3. What are the termination codons and how are they recognized?
  1. mRNA provides the code that determines the order of amino acids in the protein tRNA transports the amino acids to the ribosome to incorporate into the growing protein chain.
  2. AUG
  3. UAA, UAG, and UGA they are recognized by special proteins called release factors, which signal the end of the translation process.

Polypeptides and Proteins

When speaking of protein synthesis it is important to make a distinction between polypeptide chains and proteins. All proteins are polypeptides but not all polypeptides are proteins however, both proteins and polypeptides are composed of amino acid monomers.

The difference between a protein and a polypeptide is the form. Smaller chains of amino acids – usually less than forty – remain as single-chain strands and are called polypeptides. Larger chains must package themselves more tightly they fold into fixed structures – secondary, tertiary, and quaternary. When a polypeptide chain folds, it is called a protein.

Polypeptide chains are formed during the translation process of protein synthesis. These polypeptides may or may not fold into proteins at a later stage. However, the term ‘protein synthesis’ is used even in the scientific community and is not incorrect.

Understanding protein synthesis is easy when we imagine our DNA as a recipe book. This book lists the instructions that show a cell how to make every tiny part of every system, organ, and tissue within our bodies. All of these individual parts are polypeptides. From the keratin in your hair and fingernails to the hormones that run through your bloodstream, polypeptides and proteins are the foundation stones of every structure. Our DNA does not code for lipids or carbohydrates – it only codes for polypeptides.

The enzyme RNA polymerase opens the DNA recipe book that sits inside the cell nucleus. It uses certain pieces of code as bookmarks to find the right page. This recipe book is written in a foreign language – mRNA copies what is written without understanding it. The recipes are translated into a language that other molecules can decipher at a later stage. The translators are ribosomes and tRNA. They read the recipe and can collect the right ingredients and, in the correct order, make the finished polypeptide product.


Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption

Cell-free protein synthesis (CFPS) is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by cell extracts, which usually contain 20-30 mg/mL of proteins. In general, these cell extracts are prepared by physical disruption however, this requires technical experience and special machinery. Here, we report a method to prepare cell extracts for CFPS using a biochemical method, which disrupts cells through the combination of lysozyme treatment, osmotic shock, and freeze-thaw cycles. The resulting cell extracts showed similar features to those obtained by physical disruption, and was able to synthesize active green fluorescent proteins in the presence of appropriate chemicals to a concentration of 20 μM (0.5 mg/mL).

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. LoFT (lysozyme treatment, osmotic shock,…

Fig 1. LoFT (lysozyme treatment, osmotic shock, and freeze-thawing) cell extraction protocol.

Fig 2. Conditions of freezing and thawing…

Fig 2. Conditions of freezing and thawing for LoFT cell extraction.

Fig 3. Effect of centrifugation force on…

Fig 3. Effect of centrifugation force on LoFT cell extract.

Protein concentration (gray bars) and…

Fig 4. Essential chemicals for CFPS using…

Fig 4. Essential chemicals for CFPS using the LoFT cell extract.

Fig 5. CFPS using a linear DNA…

Fig 5. CFPS using a linear DNA template and LoFT cell extract.

Fig 6. Essential chemicals for CFPS using…

Fig 6. Essential chemicals for CFPS using LoFT cell extract after buffer exchange.


Transfer RNAs

Transfer RNAs (tRNAs) are structural RNA molecules and, depending on the species, many different types of tRNAs exist in the cytoplasm. Bacterial species typically have between 60 and 90 types. Serving as adaptors, each tRNA type binds to a specific codon on the mRNA template and adds the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. The tRNA molecule interacts with three factors: aminoacyl tRNA synthetases, ribosomes, and mRNA.

Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the single-stranded RNA molecule hydrogen bond with each other (Figure 12.13). This shape positions the amino-acid binding site, called the CCA amino acid binding end, which is a cytosine-cytosine-adenine sequence at the 3’ end of the tRNA, and the anticodon at the other end. The anticodon is a three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing.

An amino acid is added to the end of a tRNA molecule through the process of tRNA “charging,” during which each tRNA molecule is linked to its correct or cognate amino acid by a group of enzymes called aminoacyl tRNA synthetases. At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids. During this process, the amino acid is first activated by the addition of adenosine monophosphate (AMP) and then transferred to the tRNA, making it a charged tRNA, and AMP is released.

Figure 12.13. (a) After folding caused by intramolecular base pairing, a tRNA molecule has one end that contains the anticodon, which interacts with the mRNA codon, and the CCA amino acid binding end. (b) A space-filling model is helpful for visualizing the three-dimensional shape of tRNA. (c) Simplified models are useful when drawing complex processes such as protein synthesis.

  • Describe the structure and composition of the prokaryotic ribosome.
  • In what direction is the mRNA template read?
  • Describe the structure and function of a tRNA.

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Sumoylation of eIF4E activates mRNA translation

Eukaryotic translation initiation factor 4E (eIF4E) is the cap-binding protein that binds the 5' cap structure of cellular messenger RNAs (mRNAs). Despite the obligatory role of eIF4E in cap-dependent mRNA translation, how the translation activity of eIF4E is controlled remains largely undefined. Here, we report that mammalian eIF4E is regulated by SUMO1 (small ubiquitin-related modifier 1) conjugation. eIF4E sumoylation promotes the formation of the active eIF4F translation initiation complex and induces the translation of a subset of proteins that are essential for cell proliferation and preventing apoptosis. Furthermore, disruption of eIF4E sumoylation inhibits eIF4E-dependent protein translation and abrogates the oncogenic and antiapoptotic functions associated with eIF4E. These data indicate that sumoylation is a new fundamental regulatory mechanism of protein synthesis. Our findings suggest further that eIF4E sumoylation might be important in promoting human cancers.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Interplay between eIF4E phosphorylation and…

Interplay between eIF4E phosphorylation and sumoylation. ( A ) Disruption of eIF4E phosphorylation…

Formation of the active eIF4F…

Formation of the active eIF4F complex requires eIF4E SUMO1 modification. ( A )…

Functional significance of eIF4E sumoylation…

Functional significance of eIF4E sumoylation in apoptosis and oncogenic transformation. ( A )…


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Chapter 11 Synthesis and Processing of the Proteome

The attachment of amino acids to tRNAs - ‘charging’ in molecular biology jargon - is the function of the group of enzymes called aminoacyl-tRNA synthetases. The chemical reaction that results in aminoacylation occurs in two steps. An activated amino acid intermediate is first formed by reaction between the amino acid and ATP, and then the amino acid is transferred to the 3′ end of the tRNA, the link being formed between the -COOH group of the amino acid and the -OH group attached to either the 2′ or 3′ carbon on the sugar of the last nucleotide, which is always an A (Figure 11.4).

Figure 11.4 Aminoacylation of a tRNA. The result of aminoacylation by a Class II aminoacyl-tRNA synthetase is shown, the amino acid being attached via its -COOH group to the 3′-OH of the terminal nucleotide of the tRNA. A Class I aminoacyl-tRNA synthetase attaches the amino acid to the 2′-OH group.

With a few exceptions, organisms have 20 aminoacyl-tRNA synthetases, one for each amino acid. This means that groups of isoaccepting tRNAs are aminoacylated by a single enzyme. Although the basic chemical reaction is the same for each amino acid, the 20 aminoacyl-tRNA synthetases fall into two distinct groups, Class I and Class II, with several important differences between them . In particular, Class I enzymes attach the amino acid to the 2′-OH group of the terminal nucleotide of the tRNA, whereas Class II enzymes attach the amino acid to the 3′-OH group (Ibba et al., 2000).

Aminoacylation must be carried out accurately: the correct amino acid must be attached to the correct tRNA if the rules of the genetic code are to be followed during protein synthesis. It appears that an aminoacyl-tRNA synthetase has high fidelity for its tRNA, the result of an extensive interaction between the two, covering some 25 nm 2 of surface area and involving the acceptor arm and anticodon loop of the tRNA, as well as individual nucleotides in the D and TΨC arms. The interaction between enzyme and amino acid is, of necessity, less extensive, amino acids being much smaller than tRNAs, and presents greater problems with regard to specificity because several pairs of amino acids are structurally similar. Errors do therefore occur, at a very low rate for most amino acids but possibly as frequently as one aminoacylation in 80 for difficult pairs such as isoleucine and valine. Most errors are corrected by the aminoacyl-tRNA synthetase itself, by an editing process that is distinct from aminoacylation, involving different contacts with the tRNA (Hale et al., 1997 Silvian et al., 1999).

In most organisms, aminoacylation is carried out by the process just described, but a few unusual events have been documented. These include a number of instances where the aminoacyl-tRNA synthetase attaches the incorrect amino acid to atRNA, this amino acid subsequently being transformed into the correct one by a second, separate chemical reaction. This was first discovered in the bacterium Bacillus megaterium for synthesis of glutamine-tRNA Gln (i.e. glutamine attached to its tRNA). This aminoacylation is carried out by the enzyme responsible for synthesis of glutamic acid-tRNA Glu , and initially results in attachment of a glutamic acid to the tRNA Gln (Figure 11.5A). This glutamic acid is then converted to glutamine by transamidation catalyzed by a second enzyme. The same process is used by various other bacteria (although not Escherichia coli) and by the archaea. Some archaea also use transamidation to synthesize asparagine-tRNA Asn from aspartic acid-tRNA Asn (Ibba et al., 2000). In both of these cases, the amino acid that is synthesized by the modification process is one of the 20 that are specified by the genetic code. There are also two examples where the modification results in an unusual amino acid. The first example is the conversion of methionine to N-formylmethionine (Figure 11.5B), producing the special aminoacyl-tRNA used in initiation of bacterial translation . The second example occurs in both prokaryotes and eukaryotes and results in synthesis of selenocysteine, which is specified in a context-dependent manner by some 5′-UGA-3′ codons . These codons are recognized by a special tRNA SeCys , but there is no aminoacyl-tRNA synthetase that is able to attach selenocysteine to this tRNA. Instead, the tRNA is aminoacylated with a serine by the seryl-tRNA synthetase, and then modified by replacement of the -OH group of the serine with an -SeH, to give selenocysteine (Figure 11.5C Low and Berry, 1996).

Figure 11.5. Unusual types of aminoacylation. (A) In some bacteria, tRNA Gln is aminoacylated with glutamic acid, which is then converted to glutamine by transamidation. (B) The special tRNA used inin initiation of translation in bacteria is aminoacylated with methionine, which is then converted to N-formylmethionine. (C) tRNASeCys in various organisms is initially aminoacylated with serine.


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