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12.17: Steps of Translation - Biology

12.17: Steps of Translation - Biology



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As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. Here we’ll explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.

Initiation of Translation

Protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors (IFs; IF-1, IF-2, and IF-3), and a special initiator tRNA, called . The initiator tRNA interacts with the start codon AUG (or rarely, GUG), links to a formylated methionine called fMet, and can also bind IF-2. Formylated methionine is inserted by at the beginning of every polypeptide chain synthesized by E. coli, but it is usually clipped off after translation is complete. When an in-frame AUG is encountered during translation elongation, a non-formylated methionine is inserted by a regular Met-tRNAMet.

In E. coli mRNA, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. Guanosine triphosphate (GTP), which is a purine nucleotide triphosphate, acts as an energy source during translation—both at the start of elongation and during the ribosome’s translocation.

In eukaryotes, a similar initiation complex forms, comprising mRNA, the 40S small ribosomal subunit, IFs, and nucleoside triphosphates (GTP and ATP). The charged initiator tRNA, called Met-tRNAi, does not bind fMet in eukaryotes, but is distinct from other Met-tRNAs in that it can bind IFs.

Instead of depositing at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5′ end of the mRNA. A cap-binding protein (CBP) and several other IFs assist the movement of the ribosome to the 5′ cap. Once at the cap, the initiation complex tracks along the mRNA in the 5′ to 3′ direction, searching for the AUG start codon. Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case. According to Kozak’s rules, the nucleotides around the AUG indicate whether it is the correct start codon. Kozak’s rules state that the following consensus sequence must appear around the AUG of vertebrate genes: 5′-gccRccAUGG-3′. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.

Once the appropriate AUG is identified, the other proteins and CBP dissociate, and the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit. This step completes the initiation of translation in eukaryotes.

Translation, Elongation, and Termination

In prokaryotes and eukaryotes, the basics of elongation are the same, so we will review elongation from the perspective of E. coli. The 50S ribosomal subunit of E. coli consists of three compartments: 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 exception to this assembly line of tRNAs: in E. coli, is capable of entering the P site directly without first entering the A site. Similarly, the eukaryotic Met-tRNAi, with help from other proteins of the initiation complex, binds directly to the P site (Figure 1). In both cases, this creates an initiation complex with a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

During translation elongation, the mRNA template provides specificity. As the ribosome moves along the mRNA, each mRNA codon comes into register, and specific binding with the corresponding charged tRNA anticodon is ensured. If mRNA were not present in the elongation complex, the ribosome would bind tRNAs nonspecifically.

Elongation proceeds with charged tRNAs entering the A site and then shifting to the P site followed by the E site with each single-codon “step” of the ribosome. Ribosomal steps are induced by conformational changes that advance the ribosome by three bases in the 3′ direction. The energy for each step of the ribosome is donated by an elongation factor that hydrolyzes GTP. 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 enzyme that is integrated into the 50S ribosomal subunit. The energy for each peptide bond formation is derived from GTP hydrolysis, which is catalyzed by a separate elongation factor. 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 (Figure 2). 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.

Practice Questions

Many antibiotics inhibit bacterial protein synthesis. For example, tetracycline blocks the A site on the bacterial ribosome, and chloramphenicol blocks peptidyl transfer. What specific effect would you expect each of these antibiotics to have on protein synthesis?

Tetracycline would directly affect:

  1. tRNA binding to the ribosome
  2. ribosome assembly
  3. growth of the protein chain

[reveal-answer q=”10129″]Show Answer[/reveal-answer]
[hidden-answer a=”10129″]Answer a. Tetracycline would directly affect tRNA binding to the ribosome.

[/hidden-answer]

Chloramphenicol would directly affect

  1. tRNA binding to the ribosome
  2. ribosome assembly
  3. growth of the protein chain

[reveal-answer q=”10029″]Show Answer[/reveal-answer]
[hidden-answer a=”10029″]Answer c. Chloramphenicol would directly affect growth of the protein chain.[/hidden-answer]

Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.


Translation, Elongation, and Termination

In prokaryotes and eukaryotes, the basics of elongation are the same, so we will review elongation from the perspective of E. coli. The 50S ribosomal subunit of E. coli consists of three compartments: 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. Similarly, the eukaryotic Met-tRNA binds directly to the P site (Figure 1). In both cases, this creates an initiation complex with a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

Figure 1. Ribosome mRNA translation

During translation elongation, the mRNA template provides specificity. As the ribosome moves along the mRNA, each mRNA codon comes into register, and specific binding with the corresponding charged tRNA anticodon is ensured. If mRNA were not present in the elongation complex, the ribosome would bind tRNAs nonspecifically.

Elongation proceeds with charged tRNAs entering the A site and then shifting to the P site followed by the E site with each single-codon “step” of the ribosome. 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 enzyme 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 (Figure 2). 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.

Practice

Figure 2. Translation begins when an initiator tRNA anticodon recognizes a codon on mRNA. The large ribosomal subunit joins the small subunit, and a second tRNA is recruited. As the mRNA moves relative to the ribosome, the polypeptide chain is formed. Entry of a release factor into the A site terminates translation and the components dissociate.

Many antibiotics inhibit bacterial protein synthesis. For example, tetracycline blocks the A site on the bacterial ribosome, and chloramphenicol blocks peptidyl transfer. What specific effect would you expect each of these antibiotics to have on protein synthesis?

Tetracycline would directly affect:

Chloramphenicol would directly affect

Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released. The small and large ribosomal subunits dissociate from the mRNA and from each other they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.


Molecular Basis of Inheritance

1. Activation of amino acid. 2. Transfer of activated amino acid to tRNA. 3. Initiation of synthesis. 4. Elongation of polypeptide chain.

1. Activation of amino acid. In the presence of an ATP, an amino acid combines with a specific enzyme called aminoacyl tRNA synthetase. Mg ++ is required. It produces amino acyl adenylate synthetase.

2. Transfer of activated amino acid to tRNA (Charging of tRNA). The

activated amino acid is transferred to its specific tRNA. A high energy ester bond is formed between carboxylic group of amino acid and 3&rsquo hydroxylic group of terminal adenosine tRNA. tRNA has two selection sites, one for recognizing the specific aminoacyl tRNA synthetase and the other anticodon for recognizing the codon on mRNA.

3. Initiation of synthesis. mRNA becomes attached to smaller subunit. The various initiation factors required are IF-1, IF-2 and IF-3. In eukaryotes, no IF-2 is found. The attachment is such that initiation codon of mRNA (AUG or GUG) comes to he at the P-site.

Aminoacyl t-RNA complex specific for the initiation codon (methionine-tRNA, valine tRNA and formylated methionine tRNA in prokaryotes) reaches the P-site. Anticodon (UAC of met tRNA) establishes temporary hydrogen bonds with codon of mRNA The codon-anticodon reaction occurs in the presence of GTP and IF. Now the larger subunit of ribosome combines with mRNA-met tRNA complex. The A-site is exposed as such.

4. Elongation. (Polypeptide chain formation). An amino acyl tRNA complex reaches the A-site and attaches to mRNA codon next to initiation codon. It requires GTP and elongation factor (EF). A peptide bond is established between NH2 group of /RNA attached at A-site with the carboxyl group (&mdashCOOH) on the P-site in the presence of an enzyme peptidyl transferase.

In the meanwhile, the link between tRNA and amino acid at P-site breaks and free tRNA slips away. The /RNA at the A-site bears a dipeptide.

â–² Fig. 2.12. Steps of Translation.

In the next step, the t-RNA at the A-site (bearing dipeptide) is pulled to the P-site along with mRNA. In this process, the mRNA moves by one triplet thus exposing the new codon at the A-site. This process is carried by an enzyme translocase and energy from GTP and termed translocase and energy form GTP and termed translocation. Another amino acyl tRNA complex reaches the A-site and bond is formed. Only one by one codon of mRNA are exposed at A-site and get decoded through incorporation of amino acid in the peptide chain which elongates and lie in the groove of larger subunit.

5. Termination. Synthesis of polypeptide terminates when a stop signal (UAA, UAG or UGA) reaches the A-site. The P-site tRNA is hydrolysed and completed polypeptide is released in presence of releasing factors (RF). The two subunits of ribosomes also dissociates.

In case of free cytoplasmic polyribosomes, the polypeptides are released into cytoplasm when they are employed for synthesis of more cytoplasm, enzyme and components of cell organelles.


Codons

Given the different numbers of “letters” in the mRNA and protein “alphabets,” scientists theorized that combinations of nucleotides corresponded to single amino acids. Nucleotide doublets would not be sufficient to specify every amino acid because there are only 16 possible two-nucleotide combinations (42). In contrast, there are 64 possible nucleotide triplets (43), which is far more than the number of amino acids. Scientists theorized that amino acids were encoded by nucleotide triplets and that the genetic code was degenerate. In other words, a given amino acid could be encoded by more than one nucleotide triplet. This was later confirmed experimentally Francis Crick and Sydney Brenner used the chemical mutagen proflavin to insert one, two, or three nucleotides into the gene of a virus. When one or two nucleotides were inserted, protein synthesis was completely abolished. When three nucleotides were inserted, the protein was synthesized and functional. This demonstrated that three nucleotides specify each amino acid. These nucleotide triplets are called codons. The insertion of one or two nucleotides completely changed the triplet reading frame, thereby altering the message for every subsequent amino acid (Figure 5). Though insertion of three nucleotides caused an extra amino acid to be inserted during translation, the integrity of the rest of the protein was maintained.

Figure 5. The deletion of two nucleotides shifts the reading frame of an mRNA and changes the entire protein message, creating a nonfunctional protein or terminating protein synthesis altogether.

Scientists painstakingly solved the genetic code by translating synthetic mRNAs in vitro and sequencing the proteins they specified (Figure 6).

Figure 6. This figure shows the genetic code for translating each nucleotide triplet in mRNA into an amino acid or a termination signal in a nascent protein. (credit: modification of work by NIH)

In addition to instructing the addition of a specific amino acid to a polypeptide chain, three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called nonsense codons, or 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. Conservation of codons means that a purified mRNA encoding the globin protein in horses could be transferred to a tulip cell, and the tulip would synthesize horse globin. That there is only one genetic code is powerful evidence that all of life on Earth shares a common origin, especially considering that there are about 1084 possible combinations of 20 amino acids and 64 triplet codons.

Degeneracy is believed to be a cellular mechanism to reduce the negative impact of random mutations. Codons that specify the same amino acid typically only differ by one nucleotide. In addition, amino acids with chemically similar side chains are encoded by similar codons. This nuance of the genetic code ensures that a single-nucleotide substitution mutation might either specify the same amino acid but have no effect or specify a similar amino acid, preventing the protein from being rendered completely nonfunctional.


The Lac operon

In lac operon (here lac referes to lactose), a polycistronic structural gene is regulated by a common promoter and regulatory genes.

xThe lac operon consists of one regulatory gene (the i gene – here the term i does not refer to inducer, rather it is derived from the word inhibitor) and three structural genes (z, y, and a). The i gene codes for the repressor of the lac operon. The z gene codes for beta-galactosidase (ß-gal), which is primarily responsible for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose and glucose. The y gene codes for permease, which increases permeability of the cell to ß-galactosides. The a gene encodes a transacetylase. Hence, all the three gene products in lac operon are required for metabolism of lactose. In most other operons as well, the genes present in the operon are needed together to function in the same or related metabolic pathway.

Lactose is the substrate for the enzyme beta-galactosidase and it regulates switching on and off of the operon. Hence, it is termed as inducer. In the absence of a preferred carbon source such as glucose, if lactose is provided in the growth medium of the bacteria, the lactose is transported into the cells through the action of permease. The lactose then induces the operon in the following manner.

The repressor of the operon is synthesised (all-the-time – constitutively) from the i gene. The repressor protein binds to the operator region of the operon and prevents RNA polymerase from transcribing the operon. In the presence of an inducer, such as lactose or allolactose, the repressor is inactivated by interaction with the inducer. This allows RNA polymerase access to the promoter and transcription proceeds.


Translation: Synthesis of Proteins (With Diagram)

Translation is the mechanism by which the triplet base sequence of a mRNA guides the linking of a specific sequence of amino acids to form a polypeptide (protein) on ribosomes.

Protein synthesis requires amino acids, DNA, RNAs, ribosomes and enzymes. The mechanism of protein synthesis involves four steps. The four steps are: (1) Activation of Amino Acids (2) Charging of tRNA (3) Activation of Ribosomes and (4)Assembly of Amino Acids (Polypeptide Formation).

Machinery for Protein Synthesis:

Protein synthesis requires amino acids, DNA, RNAs, ribosomes and enzymes.

I. Amino Acids:

Proteins are the polymers of amino acids. Therefore, amino acids form the raw material for protein synthesis. The proteins of living organisms need about 20 amino acids as building blocks or monomers. These are available in the cytoplasmic matrix as an amino acid pool.

II. DNA as Specificity Control:

A cell, in order to maintain its own special characteristics, must manufacture proteins exactly similar to those present already in it. Thus, protein synthesis requires specificity control to provide instructions about the exact sequence in which the given numbers and kinds of amino acids should be linked to get the desired polypeptides.

The specificity control is exercised by DNA through mRNA sequences of 3 consecutive nitrogenous bases in the DNA double helix form the biochemical or genetic code. Each base triplet codes for a specific amino acid. Since the DNA is more or less stable, the proteins formed in a cell are exactly like the preexisting proteins.

III. RNAs:

RNA molecule is a long, un-branched, single-stranded polymer of ribonucleotides (Fig. 7.12). Each nucleotide unit is composed of three smaller molecules: a phosphate group, a 5- carbon ribose sugar, and a nitrogen-containing base. The bases in RNA are adenine, guanine, uracial and cytosine. The various components are linked up as in DNA.

There are three types of RNA in every cell: messenger RNA or mRNA, ribosomal RNA or rRNA and transfer RNA or tRNA. The three types of RNAs are transcribed from different regions of DNA template, RNA chain is complementary to the DNA strand which produces it. All the three kinds of RNAs play a role in protein synthesis.

The DNA, that controls protein synthesis, is located in the chromosomes within the nucleus, whereas the ribosomes, on which the protein synthesis actually occurs, are placed in the cytoplasm. Therefore, some sort of agency must exist to carry instructions from the DNA to the ribosomes. This agency does exist in the form of mRNA.

The mRNA carries the message (information) from DNA about the sequence of particular amino acids to be joined to form a polypeptide, hence its name. It is also called informational RNA or template RNA. The mRNA forms about 5% of the total RNA of a cell. Its molecule is linear and the longest of all the three RNA types. Its length is related to the size of the polypeptide to be synthesized with its information.

There is a specific mRNA for each polypeptide. Because of the variation of size in mRNA population in a cell, the mRNA is often called heterogeneous nuclear RNA, or hn RNA:

In eukaryotes, mRNA carries information for one polypeptide only It is monocistronic (monogenic) because it is transcribed from a single cistron (gene) and has a single initiator codon and a single terminator codon.

Bacterial mRNA often carries information for more than one polypeptide chains. Such a mRNA is said to be polycistronic (polygenic) because it is transcribed from many contiguous (adjacent) genes. A polycistronic mRNA has an initiator codon and a terminator codon for each polypeptide to be formed by it.

The tRNA has many varieties. Each variety carries a specific amino acid from the amino acid pool to the mRNA on the ribosomes to form a polypeptide, hence its name. The tRNAs form about 15% of the total RNA of a cell. Its’ molecule is the smallest of all the RNA types.

A tRNA molecule has the form of a clover leaf. It has four regions:

This is the 3′ end of the molecule. Here a specific amino acid joins it. It in all cases has a base triplet CCA with – OH at the tip. The – COOH of amino acid joins the – OH.

It is the opposite end of the molecule. It has 3 unpaired ribonucleotides. The bases of these ribonucleotides have complementary bases on the mRNA chain. A base triplet on mRNA chain is called a codon, and its complementary base triplet on tRNA molecule is termed an anticodon. Anticodon reads its appropriate codon and temporarily joins it by hydrogen bonds during protein synthesis.

It is on one side of the molecule. It is meant for a specific charging enzyme which catalyzes the union of a specific amino acid to tRNA molecule.

It is on the other side of the molecule. It is meant for attachment to a ribosome.

The rRNA molecule is greatly coiled. In combination with proteins, it forms the small and large subunits of the ribosomes, hence its name. It forms about 80% of the total RNA of a cell. The rRNA also seems to play some general role in protein synthesis.

(IV) Ribosomes:

Ribosomes serve as the site for protein synthesis. The small and large subunits of ribosomes occur separately when not involved in protein synthesis. The two sub units form association (join) when protein synthesis starts, and undergo dissociation (separate) when protein synthesis stops. Many ribosomes line up on the mRNA chain during protein synthesis. Such a group of active ribosomes is called a polyribosome, or simply a polysome.

In a polysome, the adjacent ribosomes are about 340 Å apart. The number of ribosomes in a polysome is related to the length of the mRNA molecule, which reflects the length of the polypeptide to be synthesized. It has been established that polypeptides are synthesized at the polysomes and not at the single free ribosomes as held earlier. This is true for both prokaryotes and eukaryotes as well as for the cell organelles such as mitochondria and plastids.

A ribosome has two binding sites for tRNA molecules. One is called A (acceptor or aminoacyl) site and the other is termed P (peptidyl) site. These sites span across the large and small subunits of the ribosome (Fig. 7.13). The A site receives the tRNA-amino acid complex. From P site, the tRNA leaves after leaving its amino acid to the forming polypeptide. However, the first tRNA-amino acid complex directly enters the P site of the ribosome.

The function of the ribosome is to hold in position the mRNA, tRNA and the associated enzymes controlling the process until a peptide bond forms between the adjacent amino acids.

Mechanism of Protein Synthesis:

Protein biosynthesis involves following major steps:

(i) Activation of Amino Acids:

Amino acid reacts with ATP to form amino acid -AMP complex and pyrophosphate. The reaction is catalyzed by a specific amino acid-activating enzyme called aminoacyl- tRNA synthetase in the presence of Mg 2+ . There is a separate aminoacyl – tRNA synthetize enzyme for each kind of amino acid. Much of the energy released by the separation of phosphate groups from ATP is trapped in the amino acid — AMP complex.

The complex remains temporarily associated with the enzyme. The amino acid-AMP-enzyme complex is called an activated amino acid (Fig. 7.14). The pyrophosphate is hydrolysed to 2Pi, driving the reaction to the right.

(ii) Charging of tRNA:

The amino acid-AMP- enzyme complex joins to the amino acid binding site of its specific tRNA, where its -COOH group bonds to – OH group of the terminal base triplet CCA. The reaction is catalyzed by the same aminoacyl-tRNA synthetase enzyme.

The resulting tRNA-amino acid complex is called a charged tRNA (Fig. 7.14). AMP and enzyme are freed. The freed enzyme can activate and attach another amino acid molecule to another tRNA molecule. The energy released by change of ATP to AMP is retained in the amino acid-tRNA complex. This energy is later used to drive the formation of peptide bond when amino acids link together on ribosomes.

The tRNA-amino acid complex moves to the site of protein synthesis, the ribosome.

(iii) Activation of Ribosomes:

The small and the large subunits of ribosomes must be joined together for protein synthesis. This is brought about by mRNA chain. The latter joins the small ribosomal subunit by first codon through base pairing with appropriate sequence on rRNA. The combination of the two is called initiation complex (Fig. 7.15). The large subunit later joins the small subunit, forming active ribosome. Activation of ribosome by mRNA requires proper concentration of Mg 2+ (0.001 Molar conc.)

(iv) Assembly of Amino Acids (Polypeptide Formation):

The events in protein synthesis are better known in bacteria than in eukaryotes. Although these are thought to be similar in the two groups, some differences do occur. The following description refers mainly to protein synthesis in bacteria on the 70S ribosomes. Polypeptide formation involves 3 events: initiation, elongation and termination of amino acid chain.

(a) Initiation of Polypeptide Chain:

The mRNA chain has at its 5′ end an “initiator” or “start” codon (AUG) that signals the start of polypeptide formation. This codon lies close to the P site of the ribosome. The amino acid formyl-methionine (methionine in eukaryotes) initiates the process. It is carried by tRNA having UAC anticodon which bonds to AUG initiator codon of mRNA by hydrogen bonds.

Initiation factors (IF 1, IF 2 and IF 3) and GTP promote the initiation process. The large ribosomal subunit now joins the small subunit to complete the ribosome. At this stage, GTP is hydrolyzed to GDP. The ribosome has formylmethionine-bearing tRNA (tRNA f Met ) at the P site (Fig. 7.15). Later, the formylmethionine is changed to normal methionine by the enzyme deformylase. If not required, methionine is later separated from the polypeptide chain by a proteolytic enzyme amino peptidase.

Initiation factors are used again to start new chains. As already established, translation of the codons of mRNA takes place in the 5′ – 3′ direction, thus P site and A site on the ribosomes recognize the polarity of the mRNA chain.

At this point fmet- tRNAf met molecule in the 70S initiation complex occupies the P site on the ribosome. The other site for a tRNA molecule, i.e. the A site, is empty. The fmet-tRNAf met is positioned in such a way that its anticodon pairs with the initiating AUG (or GUG) codon on mRNA. The reading frame is specified by this interaction and by pairing of the adjoining purine-rich sequence to a pyrimidine-rich sequence in 16S rRNA.

The elongation cycle in the protein synthesis begins with the insertion of an aminoacyl tRNA into the empty A site on the ribosome. The species of tRNA to be inserted depends upon the mRNA codon that is present in the A site. The complementary aminoacyl tRNA is transferred to the A site by a non-ribosomal specific cytoplasmic protein, called the elongation factor T (EF-T) that binds to the aminoacyl tRNA.

The factor EF-T contains two subunits, EF-Ts and EF-Tu. EF-Tu like IF2 contains a bound guanyl nucleotide and cycles between a GTP and a GDP. If the codon matches the anticodon, GTP is hydrolysed, positioning the aminoacyl tRNA in the A site and GDP bound with EF-Tu dissociates from the ribosome. A second elongation factor EF-Ts joins the EF-Tu complex and GDP is displaced from the complex forming a EF-Tu-Ts complex.

Finally, GTP binds to the EF-Tu- EF-Ts complex, releasing EF-Ts. EF-Tu containing bound GTP is ready to pick up another aminoacyl tRNA and deliver to the A site of the ribosome. This GTP-GDP cycle keeps repeating. It should be noted that EF-Tu does not recognise the fmet-tRNA initiator, hence the initiator tRNA is not delivered to the A site.

On the contrary before fmet-tRNAet, like all other aminoacyl tRNAs, can bind to EF-Tu. This explains why internal AUG codons are not read by initiator tRNA. It has been observed that rapid binding of EF-Tu to an activated aminoacyl-tRNA prevents hydrolysis, but after the formation of H’-Tu-GTP-tRNA complex, a time lag of several milliseconds allows the codon-anticodon mismatches to diffuse away (before OTP hydrolysis).

Peptide Bond Formation and Translocation:

Once the initiator fmet-tRNA occupies the P site and the next aminoacyl-tRNA occupies the A site, a peptide bond between the adjacent amino acids is formed by an enzyme, peptidyl transferase belonging to the 50S subunit. The active site of the peptidyl transferase is the 23 S rRNA. The uncharged tRNAf met occupies the P site and the dipeptide formed is attached to the second tRNA occupying the A site following the formation of a peptide bond. The product of the first peptide bond formation is called dipeptidyl-tRNA bound to the A site.

The next step of the elongation cycle is translocation, which requires a third elongation factor EF-G (also called translocase) causing hydrolysis of GTP.

Three important movements occur:

(1) The fmet-tRNA which is now uncharged leaves the P site,

(2) The second tRNA with bound dipeptide is moved to the P site, and

(3) mRNA moves a distance of three nucleotides.

After translocation, the A site is opened up to accept the incoming aminoacyl-tRNA to match the next codon, now positioned at the A site for the next round of elongation (Fig. 7.16). The factor EF-Tu delivers the next aminoacyl-tRNA for the empty A site.

The accuracy of protein synthesis depends on having the correct aminoacyl-tRNA in the A site when the peptide bond is formed, hence the incoming aminoacyl-tRNA is meticulously scrutinized so that its anticodon is complementary and matches the codon at the A site. A mismatch aminoacyl- tRNA may bind with two or three nucleotides of a codon only temporarily, but will leave the A site before a peptide bond is formed. It takes a few milliseconds for the ribosome to decide if the incoming aminoacyl-tRNA is the correct one or not and the time lag is determined by GTPase site of EF-Tu. A peptide bond cannot be formed until EF-Tu is released from the aminoacyl-tRNA and the process requires hydrolysis of GTP to GDP and Pi.

Two conditions are necessary for termination of protein synthesis. One is the presence of a stop codon that signals the chain elongation to terminate, and the other is the presence of release factors (RF) which recognise the chain terminating signal. There are three terminating codons, UAA, UGA and UAG for which tRNAs do not exist. Termination of polypeptide chain is signaled by one of these codons in the mRNA. Behind all this complexity is the fact that after the polypeptide chain has reached its full length, its carboxyl end is still bound to its tRNA adapter.

Termination must, therefore, involve the splitting of the terminal tRNA. Release of the peptidyl tRNA from the ribosome is promoted by three specific release factors, RF1, RF2 and RF3. RF1 recognises triplets UAA and UAG, while RF2 recognises UAA and UGA. The third factor RF3 does not possess any release activity of its own, but it binds to OTP and stimulates the binding of RF1 and RF2 with the ribosome.

In E. coli, 16S rRNA is essential in reading the stop codon. The release factors bind to stop codon to cause a shift of the polypeptidyl-tRNA from A to P site (Fig. 7.17). Whether OTP hydrolysis is required for chain termination is not yet firmly established, although the RF3, which appears to enhance RF1 and RF2 binding with ribosome, does not bind to OTP.

The ester bond between the polypeptide chain and the last tRNA is then hydrolysed. Binding of RF to the terminating codon causes water to act as the acceptor of the growing peptide and not another amino acid on a tRNA Release of the polypeptide chain is followed by dissociation of mRNA and tRNA. Subsequently dissociation of 30S and SOS ribosome subunits takes place with concomitant binding of IF3 to 30S subunit to prevent reassembly in the absence of mRNA and fmet-tRNA.

Modification of Released Polypeptide:

The just released polypeptide has primary structure, i.e., it is a straight, linear molecule. It is often called as nascent polypeptide. It may lose some amino acids from the end with the help of an exopeptidase enzyme, and then coil and fold on itself to acquire secondary and tertiary structure. It may combine with other polypeptides to have quaternary structure. The proteins synthesized on free polysomes are released into the cytoplasm and function as structural and enzymatic proteins. The proteins formed on the polysomes attached to ER pass into the ER channels and are exported as cell secretions by exocytosis after packaging in the Golgi apparatus.

Polysome Formation and Translational Amplification:

When the ribosome has moved sufficiently down the mRNA chain towards 3′ end, another ribosome takes up position at the initiator codon of mRNA, and starts synthesis of a second copy of the same polypeptide chain. At any given time, the mRNA chain will, therefore, carry many ribosomes over which are similar polypeptide chains of varying length, shortest near the initiator codon and longest near the stop codon.

A row of ribosomes joined to the mRNA molecule, is called a polyribosome, or simply a polysome. Synthesis of many molecules of the same polypeptide simultaneously from one mRNA molecule by a polysome is called translational amplification.

Inhibitors of Protein Synthesis in Prokaryotes:

Antibiotics are the bio-chemicals synthesized by bacteria and some fungi. Many antibiotics are known to block the bacterial translocation. This forms the basis of checking bacterial infection without harming the human host.


Watch the video: Translation (August 2022).