Prokaryotic Translation Steps, Requirements.

Prokaryotic Translation

In molecular biology and genetics, translation is the process in which ribosomes in the cytoplasm or endoplasmic reticulum synthesize proteins after the process of transcription of DNA to RNA in the cell’s nucleus. The entire process is called gene expression.

• During the translation process, the messenger RNA (mRNA) decoded within the ribosome to form a specific amino acid chain, or polypeptide.
• Later this polypeptide chain folds into an active protein and performs its functions in the cell.
• Translation involves the transfer of the information in mRNA molecules into the sequences of amino acids in polypeptide gene products.
• The process of translation in both Prokaryotic and eukaryotic cells is different. In this article we will discuss the Prokaryotic Translation.
• The Prokaryotic Translation occurs within the cytosol, where the large and small subunits of the ribosome bind to the mRNA.

• The Prokaryotic Translation completed in three distinct phases such as Initiation, Elongation, and Termination.
• During the initiation phase, the ribosome assembles around the target mRNA. The first tRNA is attached at the start codon.
• During the elongation phase, the last tRNA validated by the small ribosomal subunit (accommodation) transfers the amino acid it carries to the large ribosomal subunit which binds it to one of the preceding admitted tRNA (transpeptidation). The ribosome then moves to the next mRNA codon to continue the process (translocation), creating an amino acid chain.
• In the Termination phase, when a stop codon is reached, the ribosome releases the polypeptide. The ribosomal complex remains intact and moves on to the next mRNA to be translated.

Component Required for Prokaryotic Translation

Amino acids

• Proteins are polymers of amino acids. Of the 20 amino acids found in protein structure, half of them (10) can be synthesized by man. About 10 essential amino acids have to be provided through the diet.
• Protein synthesis can only occur when all the amino acids required for a particular protein are available. If there is a deficiency in the dietary supply of any one of the essential amino acids, the translation will stop. 
• It is, therefore, necessary that a regular dietary supply of essential amino acids, in sufficient quantities, is maintained, as it is a prerequisite for protein synthesis.
• As regards prokaryotes, there is no requirement of amino acids, since all the 20 are synthesized from the inorganic components.

Ribosomes

• The functionally active ribosomes are the centres or factories for protein synthesis. Ribosomes may also be considered as workbenches of translation.
• Ribosomes are approximately half protein and half RNA.
• Ribosomes are huge complex structures (70S for prokaryotes and 80S for eukaryotes) of proteins and ribosomal RNAs.
• Each ribosome consists of two subunits—one big and one small, which dissociate when the translation of an mRNA molecule is completed and reassociate during the initiation of translation. 
• In E. coli, the small (30S) ribosomal subunit contains a 16S (molecular weight about 6 3 105) RNA molecule plus 21 different polypeptides, and the large (50S) subunit contains two RNA molecules (5S, molecular weight about 4 3 104, and 23S, molecular weight about 1.2 3 106) plus 31 polypeptides.
• In mammalian ribosomes, the small subunit contains an 18S RNA molecule plus 33 polypeptides, and the large subunit contains three RNA molecules of sizes 5S, 5.8S, and 28S plus 49 polypeptides. In organelles, the corresponding rRNA sizes are 5S, 13S, and 21S.
• The functional ribosome has two sites—A site and P site. Each site covers both the subunits.
• A site is for binding of aminoacyl tRNA and P site is for binding peptidyl tRNA, during the course of translation. Some consider A site as acceptor site, and P site as donor site.
• In the case of eukaryotes, there is another site called exit site or E site. Thus, eukaryotes contain three sites (A, P and E) on the ribosomes.
• The ribosomes are located in the cytosomal fraction of the cell.
• They are found in association with rough endoplasmic reticulum (RER) to form clusters RER—ribosomes, where the protein synthesis occurs.
• The term polyribosome (polysome) is used when several ribosomes simultaneously translate on a single mRNA.
• The ribosomes provide many of the macromolecular components required for the translation process. 

Note: During translation procedure within the ribosome, The A or aminoacyl site binds the incoming aminoacyl-tRNA, the tRNA carrying the next amino acid to be added to the growing polypeptide chain. The P or peptidyl site binds the tRNA to which the growing polypeptide is attached. The E or exit site binds the departing uncharged tRNA.

Messenger RNA (mRNA)

• The specific information required for the synthesis of a given protein which is present on the mRNA.
• The DNA has passed on the genetic information in the form of codons to mRNA to translate into a protein sequence.
• The mRNA molecules provide the specifications for the amino acid sequences of the polypeptide gene products.

Transfer RNAs (tRNAs)

• Transfer RNAs carry the amino acids, and hand them over to the growing peptide chain.
• The amino acid is covalently bound to tRNA at the 3’-end. Each tRNA has a three nucleotide base sequence—the anticodon, which is responsible to recognize the codon (complementary bases) of mRNA for protein synthesis.
• In man, there are about 50 different tRNAs whereas in bacteria around 40 tRNAs are found. Some amino acids (particularly those with multiple codons) have more than one tRNA.
• tRNAs act as an adaptor molecule that must mediate the specification of amino acids by codons in mRNAs during protein synthesis.
• It contain a triplet nucleotide sequence, the anticodon, which is complementary to and base-pairs with the codon sequence in mRNA during translation. There are one to four tRNAs for each of the 20 amino acids.
• The tRNAs provide the adaptor molecules needed to incorporate amino acids into polypeptides in response to codons in mRNAs.

Energy sources

• Both ATP and GTP are required for the supply of energy in protein synthesis.
• Some of the reactions involve the breakdown of ATP or GTP, respectively, to AMP and GMP with the liberation of pyrophosphate. Each one of these reactions consumes two high-energy phosphates (equivalent to 2 ATP).

Protein factors

• The process of translation involves a number of protein factors.
• These are needed for initiation, elongation, and termination of protein synthesis.
• The protein factors are more complex in eukaryotes compared to prokaryotes.

Factors	Translation steps	Functions
IF-1	Initiation	Helps to stabilize 30S ribosomal subunit
IF-2	Initiation	Binds fmet-tRNA with 30S subunit mRNA complex; bind GTP and hydrolyse
IF-3	Initiation	Binds 30S subunit with mRNA
EF-TU	Elongation	Binds GTP; bring Aminoacyl-tRNA to A-site of ribosome
EF-TS	Elongation	Generates EF-TU
EF-G	Elongation	Helps in translocation of ribosome
RF-1	Termination	Helps to dissociates polypeptide from tRNA ribosome complex; specific for UAA and UAG
RF-2	Termination	Helps to dissociates polypeptide; specific for UGA and UAA
RF-3	Termination	Stimulates RF-1 and RF-2

Activation of Amino acid/Synthesis of aminoacyl-tRNA

• Amino acids are activated and attached to tRNAs in a two-step reaction. This reaction is take place in the cytosol.
• A group of enzymes namely aminoacyl tRNA synthetases is required for this process.
• These enzymes are highly specific for the amino acid and the corresponding tRNA.
• The amino acid is first attached to the enzyme utilizing ATP to form enzyme-AMP-amino acid complex. The amino acid is then transferred to the 3’ end of the tRNA to form aminoacyl tRNA.
• The amino acids are attached to the tRNAs by high-energy (very reactive) bonds (symbolized ~) between the carboxyl groups of the amino acids and the 3-hydroxyl termini of the tRNAs
• The aminoacyl~tRNAs are the substrates for polypeptide synthesis on ribosomes, with each activated tRNA recognizing the correct mRNA codon and presenting the amino acid in a steric confi guration (three-dimensional structure) that facilitates peptide bond formation.
• The linkage of an amino acid to a tRNA is crucial for two reasons. First, the attachment of a given amino acid to a particular tRNA establishes the genetic code.When an amino acid has been linked to a tRNA, it will be incorporated into a growing polypeptide chain at a position dictated by the anticodon of the tRNA.
• Second, because the formation of a peptide bond between free amino acids is not thermodynamically favorable, the amino acid must first be activated for protein synthesis to proceed. The activated intermediates in protein synthesis are amino acid esters, in which the carboxyl group of an amino acid is linked to either the – or the -hydroxyl group of the ribose unit at the end of tRNA.
• An amino acid ester of tRNA is called an aminoacyl-tRNA or sometimes a charged tRNA.

Reaction

• The activation reaction is catalyzed by specific aminoacyl-tRNA synthetases, which are also called activating enzymes. The first step is the formation of an aminoacyl adenylate from an amino acid and ATP.

Amino acid + ATP ⇄ aminoacyl-AMP + PPi

• This activated species is a mixed anhydride in which the carboxyl group of the amino acid is linked to the phosphoryl group of AMP; hence, it is also known as aminoacyl-AMP.
• The next step is the transfer of the aminoacyl group of aminoacyl-AMP to a particular tRNA molecule to form aminoacyl-tRNA.

Aminoacyl-AMP + tRNA ⇄ aminoacyl-tRNA + AMP

The sum of these activation and transfer steps is

Amino acid + ATP + tRNA ⇄ aminoacyl-tRNA + AMP + PPi

The of this reaction is close to 0, because the free energy of hydrolysis of the ester bond of aminoacyl-tRNA is similar to that for the hydrolysis of ATP to AMP and PPi. As we have seen many times, the reaction is driven by the hydrolysis of pyrophosphate. The sum of these three reactions is highly exergonic:

Amino acid + ATP + tRNA + H2O → aminoacyl-tRNA + AMP + 2Pi

• Thus, the equivalent of two molecules of ATP is consumed in the synthesis of each aminoacyl-tRNA. One of them is consumed in forming the ester linkage of aminoacyl-tRNA, whereas the other is consumed in driving the reaction forward.
• The activation and transfer steps for a particular amino acid are catalyzed by the same aminoacyl-tRNA synthetase. Indeed, the aminoacylAMP intermediate does not dissociate from the synthetase. Rather, it is tightly bound to the active site of the enzyme by noncovalent interactions.
• Aminoacyl-AMP is normally a transient intermediate in the synthesis of aminoacyl-tRNA, but it is relatively stable and readily isolated if tRNA is absent from the reaction mixture.

Steps of Prokaryotic Translation

The Prokaryotic Translation is completed in three different steps such as;

1. Polypeptide chain initiation.
2. Chain elongation.
3. Chain termination.

Polypeptide chain Initiation

• In E. coli, the initiation process involves the 30S subunit of the ribosome, a special initiator tRNA, an mRNA molecule, three soluble protein initiation factors: IF-1, IF-2, and IF-3, and one molecule of GTP. 
• Translation occurs on 70S ribosomes, but the ribosomes dissociate into their 30S and 50S subunits each time they complete the synthesis of a polypeptide chain. 
• In the first stage of the initiation of translation, a free 30S subunit interacts with an mRNA molecule and the initiation factors. 
• The 50S subunit joins the complex to form the 70S ribosome in the final step of the initiation process.

Steps Involve in Polypeptide chain Initiation

Step One: Formation of f IF-2/tRNAMet and IF-3/mRNA/30S subunit complexes

1. Polypeptide chain initiation begins with the formation of two complexes: (a) one contains initiation factor IF-2 and methionyl-tRNAf Met, and (b) the other contains an mRNA molecule, a 30S ribosomal subunit and initiation factor IF-3. 
2. The 30S subunit/mRNA complex will form only in the presence of IF-3; thus, IF-3 controls the ability of the 30S subunit to begin the initiation process.
3. The formation of the 30S subunit/mRNA complex depends in part on base-pairing between a nucleotide sequence near the 3 end of the 16S rRNA and a sequence near the 5 end of the mRNA molecule.
4. Prokaryotic mRNAs contain a conserved polypurine tract, consensus AGGAGG, located about seven nucleotides upstream from the AUG initiation codon. This conserved hexamer, called the Shine-Dalgarno sequence after the scientists who discovered it, is complementary to a sequence near the 3 terminus of the 16S ribosomal RNA.
5. When the Shine-Dalgarno sequences of mRNAs are experimentally modified so that they can no longer base-pair with the 16S rRNA, the modified mRNAs either are not translated or are translated very inefficiently, indicating that this base-pairing plays an important role in translation.

Step Two: Formation of Initiation Complex

1. The IF-2/methionyl-tRNAf Met complex and the mRNA/30S subunit/IF-3 complex subsequently combine with each other and with initiation factor IF-1 and one molecule of GTP to form the complete 30S initiation complex.
2. The final step in the initiation of translation is the addition of the 50S subunit to the 30S initiation complex to produce the complete 70S ribosome.
3. Initiation factor IF-3 must be released from the complex before the 50S subunit can join the complex; IF-3 and the 50S subunit are never found to be associated with the 30S subunit at the same time.
4. The addition of the 50S subunit requires energy from GTP and the release of initiation factors IF-1 and IF-2.

Step three: Formation of 70S ribosome

1. The addition of the 50S ribosomal subunit to the complex positions the initiator tRNA, methionyl-tRNAf Met, in the peptidyl (P) site with the anticodon of the tRNA aligned with the AUG initiation codon of the mRNA.
2. Methionyl-tRNAf Met is the only aminoacyl-tRNA that can enter the P site directly, without first passing through the aminoacyl (A) site.
3. With the initiator AUG positioned in the P site, the second codon of the mRNA is in register with the A site, dictating the aminoacyl-tRNA binding specificity at that site and setting the stage for the second phase in polypeptide synthesis, chain elongation.

Polypeptide Chain Elongation

The process of polypeptide chain elongation is basically the same in both prokaryotes and eukaryotes. The addition of each amino acid to the growing polypeptide occurs in three steps: 

1. Step 1: binding of an aminoacyl-tRNA to the A site of the ribosome
2. Step 2: transfer of the growing polypeptide chain from the tRNA in the P site to the tRNA in the A site by the formation of a new peptide bond,
3. Step 3: translocation of the ribosome along the mRNA to position the next codon in the A site.

During step 3, the nascent polypeptide-tRNA and the uncharged tRNA are translocated from the A and P sites to the P and E sites, respectively. These three steps are repeated in a cyclic manner throughout the elongation process. The soluble factors involved in chain elongation in E. coli are described here. Similar factors participate in chain elongation in eukaryotes.

Steps Involve in Polypeptide Chain Elongation

Step One: Binding of an aminoacyl-tRNA to the A site of the ribosome

• In the first step, an aminoacyl-tRNA enters and becomes bound to the A site of the ribosome, with the specificity provided by the mRNA codon in register with the A site.
• The three nucleotides in the anticodon of the incoming aminoacyl-tRNA must pair with the nucleotides of the mRNA codon present at the A site.
• This step requires elongation factor Tu carrying a molecule of GTP (EF-Tu.GTP).
• The GTP is required for aminoacyl-tRNA binding at the A site but is not cleaved until the peptide bond is formed.
• After the cleavage of GTP, EF-Tu.GDP is released from the ribosome.
• EF-Tu.GDP is inactive and will not bind to aminoacyl-tRNAs.
• EF-Tu.GDP is converted to the active EF-Tu.GTP form by elongation factor Ts (EF-Ts), which hydrolyzes one molecule of GTP in the process.
• EF-Tu interacts with all of the aminoacyl-tRNAs except methionyl-tRNA.

Step Two: Transfer of the growing polypeptide chain from the tRNA in the P site to the tRNA in the A site by the formation of a new peptide bond

1. The second step in chain elongation is the formation of a peptide bond between the amino group of the aminoacyl-tRNA in the A site and the carboxyl terminus of the growing polypeptide chain attached to the tRNA in the P site.
2. This uncouples the growing chain from the tRNA in the P site and covalently joins the chain to the tRNA in the A site.
3. This key reaction is catalyzed by peptidyl transferase, an enzymatic activity built into the 50S subunit of the ribosome.
4. We should note that the peptidyl transferase activity resides in the 23S rRNA molecule rather than in a ribosomal protein, perhaps another relic of an early RNA-based world.
5. Peptide bond formation requires the hydrolysis of the molecule of GTP brought to the ribosome by EF-Tu in step 1.

Step Three: translocation of the ribosome along the mRNA to position the next codon in the A site.

1. During the third step in chain elongation, the peptidyl-tRNA present in the A site of the ribosome is translocated to the P site, and the uncharged tRNA in the P site is translocated to the E site, as the ribosome moves three nucleotides toward the 3 end of the mRNA molecule.
2. The translocation step requires GTP and elongation factor G (EF-G).
3. The ribosome undergoes changes in conformation during the translocation process, suggesting that it may shuttle along the mRNA molecule.
4. The energy for the movement of the ribosome is provided by the hydrolysis of GTP. 
5. The translocation of the peptidyl-tRNA from the A site to the P site leaves the A site unoccupied and the ribosome ready to begin the next cycle of chain elongation.

Polypeptide Chain Termination

Polypeptide chain elongation undergoes termination when any of three chain-termination codons (UAA, UAG, or UGA) enters the A site on the ribosome. These three stop codons are recognized by soluble proteins called release factors (RFs). In E. coli, there are two release factors, RF-1 and RF-2. RF-1 recognizes termination codons UAA and UAG; RF-2 recognizes UAA and UGA.

The termination of Polypeptide chain involve this following steps;

1. Release factor 1 binds to the UAG termination codon in the A site of the ribosome and tRNAPhe leaves the E site.
2. Release of the nascent polypeptide and RF-1 and transfer of tRNAGly from the P site to the E site.
3. Dissociation of the mRNA-tRNA-ribosome complex.

Components required for protein synthesis in bacterial cells

References

• Principles of Genetics, D. Peter Snustad, Michael J. Simmons.
• Biochemistry, Dr. U. Satyanarayana, Dr. U. Chakrapani.
• https://en.wikipedia.org/wiki/Translation_(biology)
• https://www.onlinebiologynotes.com/translation-in-prokaryotes/
• https://www.biologydiscussion.com/cell/prokaryotes/translation-in-prokaryotes-genetics/38022