Synthesis of Proteins  (Translation):

The message coded by DNA on m_ RNA is translated into a specific  protein is called protein synthesis  or translation. Its site is ribosomes  present on the endoplasmic reticulum. 

What is important in proteins synthesis?

Machinery for Protein Synthesis:

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

I) Amino Acids  as Raw Materials: Proteins are the polymers of amino acids. Therefore, amino acids are a basic requirement for protein synthesis. The proteins of living organisms need about 20  amino acids which are available as an amino acid pool in the cytoplasm.

II) DNA as Specificity Control: A cell must manufacture proteins which are exactly the same as those already present  in it. Thus, protein synthesis requires specific instructions about the exact sequence in which the given number and kinds of amino acids should be linked to get the desired polypeptides. This specificity is exercised by DNA through mRNA. Sequences of nitrogenous base triplets ( each triplet= specific amino acid) in the DNA double helix form the biochemical or genetic code. Since the DNA is more or less stable, the proteins formed in a cell are exactly like the preexisting  proteins. 

III) RNA as Intermediaries: RNAs serve as the intermediaries between DNA and proteins in a cell. mRNA brings coded information from DNA and takes part in translation. A translation unit in mRNA is the sequence of RNA that is flanked by the start codon ( AUG) sequences that are not translated and are referred to as untranslated regions ( UTR). The UTRs are present at both 5' _ end ( before start codon) and at 3' end ( after stop codon). They are required for efficient translation process. The tRNA picks up specific amino acids and takes them to the mRNA over particular codons which correspond to the anticodon on the tRNA.

What organelles synthesizes  proteins?

IV): Ribosomes as Protein Factories: Ribosomes are small ribonucleoprotein particles without a covering membrane. They are the site for protein synthesis,  thus called protein factories .Each ribosome consists of large and small subunits. The subunits of ribosomes occur separately when not involved in protein synthesis. The larger subunits is dome shaped. The two subunits associate (join)when protein synthesis starts, and undergo dissociation (separate) when protein synthesis stops . Association of ribosomes requires Mg²+. Many ribosomes line up on the mRNA chain during protein synthesis. Such a row of active ribosomes is called  a polyribosome, or simply a polysome. In a polysome, the adjacent ribosomes are about 340 A° or 34nm 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. Polypeptide are synthesized at the polysomes and not at the single free ribosomes in both prokaryotes and eukaryotes as well as in cell organelles such as mitochondria and plastids. 

  A ribosomes has one binding site for mRNA and three binding sites for tRNA: 

(i) P Site (Donor site, peptidyl _ tRNA site) that holds the tRNA carrying the growing polypeptide chain,

(ii) A Site ( aminoacyl tRNA site, Acceptet site) which holds the tRNA carrying next amino acid to be added to the chain,

(iii) Recently discovered E site (exit site) where discharged tRNAs leave the ribosomes. These sites span across  both the subunits of the ribosome.However, the first tRNA _ amino acid complex directly enters the Psite  of the ribosome.

   A eukaryotic ribosomes has a groove at the junction of the two subunits from where a tunnel extends through the large subunit and opens into a canal of the endoplasmic reticulum. The polypeptides are synthesized in the groove between the two ribosomal subunits and pass through the tunnel of the large subunit into the endoplasmic reticulum which protects it from cellular enzymes. The small subunit is ellipsoidal shaped and forms a cap over the large subunit. The  large subunit attaches to the endoplasmic reticulum by two glycoproteins named ribophorin I and II.

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

What are proteins made of?

Mechanism of Protein synthesis:

1): Activation of Amino Acids: Amino acid reacts with ATP to form amino acid _ AMP complex and pyrophosphate. The reaction is catalyzed by an enzyme aminoacyl_ tRNA synthetase in the presence of Mg²+ . There is a separate aminoacyl _ tRNA synthetase enzyme for each of the 20 amino acids. The amino acid _ AMP_ enzyme complex is called an activated amino acid. The energy made available to amino acid during its activation is later used in formation of peptide bonds.

2): Charging of  tRNA or Aminoacylation of tRNA: The amino acid AMP_ enzyme complex joins to the amino acid binding site of its specific or cognate tRNA, where its __COOH group binds to __ OH group of the terminal base triple CCA. The reaction is catalyzed by the same aminoacyl _ tRNA synthetase enzyme. The resulting tRNA_ amino acid complex is called a charged tRNA .AMP and enzyme are freed.The tRNA_ amino acid complex moves to the site of protein synthesis, the ribosome.

3): Activation of Ribosomes: The small and the large subunits of ribosomes must come together for protein synthesis. This is brought about by  mRNA chain. The mRNA first join the small ribosomal subunit by first codon through base pairing with appropriate sequence on rRNA. This combination is called initiation complex. The large subunit joins the small subunit later on forming  active ribosome. Activation of ribosome by  mRNA requires proper concentration of Mg²+ . The ribosome also acts as a catalyst ( 23S rRNA in bacteria is the enzyme _ ribozyme) for the formation of peptide bond.

4): Assembly of Amino Acids  ( Polypeptide Formation): Polypeptide formation involves 3 stages: initiation, elongation and termination of amino acid chain.

a): Initiation of Polypeptide chain :There is formation of a complex comprising of an mRNA, a tRNA bearing the first amino acid of the polypeptide, and the two subunits of a ribosomes. First, small subunit of  ribosome attaches to mRNA and a specific charged initiator tRNA. The ribosomal subunit binds to the leader segment at the 5' end of the mRNA . Synthesis of a proteins starts  with the same amino acid: methionine. The signal for initiation a polypeptide chain is the special initiation codon on mRNA. Usually the initiating codon is AUG,  but in bacteria, GUG or UUG are  also used.

  Within  10 bases upstream of AUG ( initiation codon) is a sequence that corresponds to a part or all of the hexamwe 5' ... AGGAGG....3'. This polypurine stretch is known as Shine__ Dalgarno sequence. In prokaryotes it is through that this Shine Dalgarno sequence pairs with its complement in rRNA during mRNA_ ribosome binding.

In eukaryotes, the 5'cap first tells the small subunit to bind to the mRNA's 5' end. This cap has nucleotides complementary to the nucleotides present at the 3' end of its r_ RNA. The initiation codon codes for the amino acid formylmethionine ( formyl_ met) in prokaryotes and non_ formylated methionine (met) in eukaryotes. Then the large subunit of ribosome join the small subunit completing the translation  initiation complex.At this stage, initiator aminoacyl tRNA complex specific for the initiation codon lies at the P site of the ribosome, and the A site is vacant to let another charged tRNA to enter.

The meaning of AUG and GUG codon depends on their location. If present as the first codon they are both read by the initiation reaction as formly_ methionine. But, if present within the coding region the AUG represents the amino acid methionine and GUG represents the amino acid valine.
[ The tRNA responsible for recognizing AUG codons in internal location ( for methionine) is different from tRNA recognizing AUG codon as initiation codon ( for formyl_ methionine). In eukaryotes also, there are two different tRNAs for carrying methionine at the initiation site and at internal locations].

  Proteins called initiation factors are necessary to bring all the components of the translation initiation complex together. Initiation factors are designated as IFs in prokaryotes and eIFs in eukaryotes

There are 3 initiation factors in prokaryotes IF3, IF2 and IF1. 

Eucaryotes have 9 initiation factors__ eIF2, eIF3, eIF1, eIF4A, eIF4B, eIF4C, eIF4D, eIF5, eIF6. 
In prokaryotes,  these factors are found only on 30S subunits and they are released when the 30S subunit associate with 50S subunits to generate 70S ribosomes.  Out of these IF3 or eIF2 factors join the small subunit of ribosome at the start of the process and leave when large subunit of ribosome joins small subunit. In other words, it is needed for 30S subunit to bind specifically to initiation site in mRNA.The anticodon of  aminoacyl tRNA complex establishes temporary hydrogen bonds with the initiation codon of mRNA in the presence of eIF3 in eukaryotes and IF2 in prokaryotes. 

40S_m+ tRNA met➡️➡️➡️➡️40S_ mRNA                                                                 _tRNA met

When the large subunit of ribosome attaches itself with the 40S _ mRNA_ tRNAmet.Complex to form the intact ribosome,  the initiation factors eIF1, eIF4, ( A,B,C ) in eukaryotes and IF1 in  prokaryotes are required.The end of the tRNA that carries an amino acid is located on the large subunit , while the anticodon at the other end interacts with the mRNA bound by the small subunit. So the P and A sites each extend across both ribosomal subunits. 

The energy for the joining of larger subunit is supplied  by GTP.

The formylmethionine is charged to normal methionine by the enzyme  deformylase. If not required,  methionine is later separated from the polypeptide chain by a proteolytic enzyme  amino_ peptidase.This removal reaction occurs, probably when the nascent polypeptide chain has reached a length of 15 amino acids. 

(b)Elongation of Polypeptide Chain: In this stage of translation, amino acids are added one by one to the first amino acid ( formylmethionine or methionine). Addition of an amino acid is the most rapid step in protein synthesis. 

At each addition of a new amino acid certain proteins called elongation factors are required. In prokaryotes,  the elongation factors are termed EF_ Tu, EF_ Ts and EF_ G. Elongation factor ( EF_ Tu) seems to be the most abundant protein in E.coil. There is a more complex set elongation factors in eukaryotes ( eEFI). Addition of an amino acid occurs in the following  3 cyclic steps.

(i) Codon Recognition : The charged tRNA molecule along with its amino acid e.g., proline, enters the ribosome at the A site. Its entry is mediated by EF_ Tu. A tertiary complex of EF.GTP and aminoacyl tRNA is the charged tRNA. Its anticodon GGA locates and attaches to the complementary codon CCU of mRNA chain by hydrogen bonds. GTP is hydrolyzed in this step.

(ii) Peptide Bond Formation: The amino acid ( formylmethionine/ methionine) on tRNA at P site and the newly arrived amino acid ( proline) on tRNA at A site join by a peptide bond. The reaction occurs spontaneously without the input of external energy. The reaction  is catalyzed by enzyme peptidyl transferase of the larger sub unit of the ribosome ( 23S rRNA in prokaryotes and 28S rRNA in eukaryotes _ thus peptidyl transferase is an RNA enzyme). In peptide bond formation, the linkage between the first amino acid and its tRNA is broken, and the COOH  group now forms  a peptide bond with the free NH2 group of the second amino acid. Thus, the second tRNA carries a dipeptide, formylmethionine/ methionine _ proline,  and the  first tRNA is without amino acid. 


(iii) Translocation:

This is the process in which tRNA at A site, carrying  a dipeptide, moves to the P site. It requires a factor translocase ( EF_ G) in porocaryotes and eEF2 in eukaryotes) and energy which is provided by hydrolysis of GTP. As the tRNA translocates to P site, its anticodon keeps hydrogen bonded to the mRNA codon, so that mRNA moves along with it and brings its next codon to the A site. While this happens, the  unchanges tRNA shifts from the P site to the E site, and then leaves the ribosome.  In eukaryotes the tRNA is expelled directly into the cytosol.

The mRNA moves through the ribosome in one direction only, i.e.,5' end first. This means that the ribosome moves in 5' ➡️3' direction on the mRNA. 

Now the next cycle starts. A third tRNA molecule with its own specific amino acid, e.g., arginine,  arrives at the A site of the ribosome and joins by its anticodon AGA to the complementary codon UCU of the mRNA chain. The dipeptide formylmethionine _ proline is shifted from the preceding tRNA on the third tRNA where it attaches to the amino acid arginine again with the help of peptidyl transferase ribosome. The dipeptide has now become a tripeptide, formylmethionine _ proline_ arginine. The second  tRNA, being now uncharged, shifts from the P site to the E site and then leaves the ribosome. The tRNA _ tripeptide complex is translocated from A site to P site, thus repeating  another cycle. 

As the ribosome moves over the mRNA, all the codons of mRNA arrive at the A site one after another,  and the peptide  chain lengthens. Thus, the amino acids are linked up into a polypeptide  in a sequence communicated by the DNA through the mRNA. A polypeptide chain in  the process of synthesis is often called a nascent polypeptide. 

The growing polypeptide chain always remains attached to its original ribosomes  and is not transferred from one ribosome to another. Only one polypeptide chain can be synthesized at a time on one ribosome. 

(C)Termination and Release  of Polypeptide chain: 

At  the end of mRNA chain there is a " stop", or " terminator" codon UAA ( ochre), UAG ( amber) or UGA ( opal). It reaches the A site but is not read and joined by the anticodon of any tRNA_ amino acid  complex because none of them are represented by a  tRNA .There can be no further addition of amino acids to the polypeptide  chain .Since the reaction of termination does not depend on codon_ anticodon recognition,  there seems to be no particular reason why it should require a triplet sequence. Presumably this reflects the evolution of the genetic code. In bacterial genes,  UAA is the  most  commonly used termination codon.A protein called release factor (only one eRF1 in eukaryotes and two RF1 and RF2 in procaryotes) joins the stop codon in the A site to add a water molecule to the polypeptide chain. This hydrolyses ( cleaves) the completed polypeptide from the tRNA that is at the P site. In prokaryotes RF1 is specific for UAG and UAA; RF2 specific for UAA and UGA. The polypeptide is now free from the ribosome. The ribosome jumps off the mRNA chain at the " stop" codon and dissociates into its  two subunits in the presence of dissociation factor ( DF).

The ribosomes and the tRNAs on release from the mRNA can function again in the formation of anothe polypeptide. 

5): Modifications of Released Polypeptide:

The just released polypeptide has primary  structure,i.e., it is a straight, linear molecule. It may get associated with other polypeptides to form a ß_ pleated structure and further coil and fold on itself to acquire tertiary and quaternary structure. 

A gene determines the primary structure of a protein,  and the primary structure,  in turn, determines the conformation.  Chaperons protein may help polypeptide fold correctly. 

What does proteins synthesis occur?

Proteins for Use Inside and Outside the cell: 

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 either pass into their lumen or become integrated into the membranes. The proteins released the lumen reach the Golgi apparatus for modifications like formation of hydrolytic enzymes and glycosylation.The modified proteins are packed in  vesicles for export or for formation of lysosomes, cell wall enzymes,  plasma membrane etc.

Polysome Formation:

Once the first ribosome has moved adequately down the mRNA towards 3' end, another ribosome comes to 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 therefore, carries many ribosomes over which there are similar  polypeptide chains of different lengths, shortest near the initiator codon and longest near the stop codon. This row of ribosomes joined to the mRNA molecule is called a polyribosome,  or simply a polysome. The synthesis of many molecules of the same polypeptide simultaneously from one mRNA molecules by a polysome is called translational amplification. 

Energy Used:

(i): One GTP is hydrolysed to GDP as each successive amino acid _ tRNA comes attaches to the  A site of the ribosome.

(ii) A second GTP is broken down to GDP as the ribosome moves to each new codon in the mRNA.

(iii) One ATP is hydrolysed to AMP during amino acid activation.

Thus, the formation of each peptide bond uses 3 high_ energy molecules, one ATP and two GTP.

                    Formation of
ATP+ 2GTP➡️➡️➡️➡️➡️AMP + 2GDP + 4H2PO

Location: Location of DNA in nucleus and ribosomes in cytoplasm of a cell is advantageous. If ribosomes were in the nucleus they would be far away from the energy source and raw materials which they required for protein synthesis. On the other hand if DNA were in the cytoplasm ,it would be  exposed to respiratory breakdown. In the nucleus, the nuclear envelope preserves stability of the DNA by protecting it form destruction by respiratory enzymes. 

Some antibiotics that interfere with protein synthesis:__

☆ Tetracycline: They bind to the 30S ribosomes subunit and inhibit binding of aminoacyl tRNA to mRNA_ ribosome complex

☆ Gentamycin and Streptomycin] : They can bind to both 30S and 50S subunits of ribosome.Inhibit initiation of translation, prevent polysome formation and also cause misreading of codon leading to abnormal polypeptide formation. 

☆Chloramphenicol: Inhibits peptidyl transferase and so formation of peptide bond between the elongating peptide chain and the newly attached aminoachyl tRNA doesn't take place.

☆ Clindamycin & Erythromycin: Bind to 50S subunit of ribosome and inhibit translocation so that the ribosome fails to move on the mRNA to expose the next codon.

☆ Neomycin: Inhibits interaction between  tRNA and mRNA.

What is an example of a proteins synthesis?

An example of protein synthesis is the process by which the instructions in a DNA molecule are used to build a protein. This involves two main steps: transcription and translation.

Transcription: During transcription, the DNA molecule unwinds and one strand is used as a template to produce a complementary RNA molecule. This occurs in the nucleus of eukaryotic cells.

Translation: After transcription, the RNA molecule, called messenger RNA (mRNA), moves from the nucleus to the cytoplasm. In the cytoplasm, ribosomes read the mRNA sequence and build a protein molecule accordingly. This involves the assembly of amino acids in the correct order based on the information carried by the mRNA. Transfer RNA (tRNA) molecules, which have an anticodon that matches specific codons on the mRNA, bring the corresponding amino acid to the ribosome. The ribosome then catalyzes the formation of peptide bonds between the amino acids, ultimately resulting in a completed protein.

Overall, this process exemplifies protein synthesis, where DNA is transcribed into mRNA, and mRNA is translated into a protein.


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