Prokaryotic Translation (Protein Synthesis) Overview

• Translation involves translating the pattern of a messenger RNA (mRNA) molecule into a sequence of amino acids during protein synthesis.
• Following the transcription of DNA into RNA, ribosomes in the cytoplasm or ER are responsible for protein synthesis.

The Ribosomes

• Normally, ribosomes exist as distinct subunits made up of rRNA and protein.
• When the subunits attach to an mRNA around its 5′ end, the ribosome is formed.
• The ribosome reads the nucleotide sequence from 5′ to 3′ in the direction of an mRNA upon binding. Then it proceeds to synthesize the appropriate protein from amino acids in an N-terminal (amino-terminal) to C-terminal (carboxyl terminal) direction.
• In the cytoplasm, ribosomes can either be attached to the endoplasmic reticulum or float freely.
• They produce proteins via synthesis.

Ribosomal Sites for Protein Translation

The schematic representation shows three tRNA binding sites on each bacterial ribosome.

1. The aminoacyl-tRNA binding siteThe entering aminoacyl-tRNA binds to the aminoacyl-tRNA binding site (also known as the A site) during elongation.
2. The peptidyl-tRNA binding site –The tRNA connected to the extending polypeptide chain is attached at the peptidyl-tRNA binding site (often called the P site).
3. The exit site – Prior to getting liberated from the ribosome, the exit site (also referred to as the E site) serves as a binding site for the transfer of RNA (tRNA)

All three locations on the ribosome are formed by rRNA molecules (A, P, and E).

THE PROCESS OF TRANSLATION

There are three steps to protein synthesis (also known as translation):

1. Initiation
2. Elongation and
3. Termination.

• At commencement, the mRNA-ribosome complex is formed, and the first codon (normally AUG) links the primary aminoacyl-tRNA (called initiator tRNA).
• In the elongation phase, new codons are successively read, and the polypeptide is prolonged by introducing amino acids toward its C-terminal end.
• This process will continue until a termination codon (stop codon) that lacks a suitable aminoacyl-tRNA to base pair is encountered.
• Now that the polypeptide has been synthesized, the ribosome releases it, and protein production is complete (termination phase).

Synthesis of aminoacyl-tRNA

Two factors make aminoacyl-tRNA synthesis extremely important:

1. Each amino acid must be covalently bonded to a tRNA molecule for protein synthesis, which relies on the tRNA’s “adaptor” activity to ensure that the proper amino acids are incorporated.
2. Due to the high-energy covalent connection formed between both the amino acid and the tRNA, the amino acid might interact with the growing polypeptide chain to create a fresh peptide bond.

• For this reason, amino acid activation is another name for the process of creating aminoacyl-tRNA.
• The anticodon is available at the end of the anticodon stem loop in the secondary structure of each cloverleaf tRNA molecule.
• At the synthesis of aminoacyl-tRNA, the amino acid is covalently bonded to the A residue of the CCA sequence at the 3′ end.
• Only one amino acid is carried by each tRNA molecule.
• The enzyme aminoacyl-tRNA synthetase catalyzes the attachment of an amino acid to a tRNA.
• There are 20 different aminoacyl-tRNA synthetases in total, one for each amino acid.

The synthesis reaction is a two-step process.

1. An aminoacyl-adenylate is created in the first stage by the reaction of an amino acid with ATP (also known as aminoacyl-AMP).
2. In the second step, the aminoacyl group of aminoacyl-AMP is moved to the 3′ end of tRNA without quitting the enzyme to form aminoacyl-tRNA.

The overall reaction is:

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

Initiation of Protein Synthesis

• The start codon, also known as the initiation codon, AUG, which codes for methionine, is the first codon translated in all mRNAs.
• tRNAfMet, also known as the initiator tRNA, is utilized for the initiation codon and is used for internal AUG codons, whereas tRNAm Met is used for both types of AUG codons.
• In prokaryotes, the first amino acid of a novel protein is N-formylmethionine (abbreviated fMet). Consequently, the aminoacyl-tRNA utilized in initiation is fMet-tRNAfMet.
• The Shine-Dalgarno region, a brief purine-rich sequence located 5′ to the AUG initiation codon, complements a portion of the 16S rRNA in the small ribosomal subunit.
• Since this is where the 30S ribosomal subunit binds, it moves along the mRNA in a 3′ orientation until it comes across the AUG initiation codon.
• Initiation factors are proteins required to begin protein synthesis (IFs).
• Three initiation factors (IF-1, IF-2, and IF-3) are essential in prokaryotes.
• The precise order of binding of IF-1, IF-2, IF-3, and fMet-tRNAf is debatable due to the intricacy of the process.

Steps Involved

1. The small (30S) ribosomal subunit is the site where IF-1 and IF-3 bind to start the reaction.

• In order to prevent a non-functional ribosome from forming in the absence of mRNA and fMet-tRNAf Met, they prevent the 30S subunit from binding to the 50S subunit.

2. The small subunit then proceeds 3′ down the mRNA in search of the AUG initiation codon after binding to the mRNA via the Shine-Dalgarno motif.
3. The initiator tRNA (fMet-tRNAfMet/IF-2/GTP) now attaches after being charged with N-formylmethionine and forming a complex with IF-2 and GTP.
4. IF-3 was released.
5. The 30S initiation complex is made up of the 30S ribosomal subunit, mRNA, fMet-tRNAf Met, IF-1, and IF-2.
6. During the liberation of IF-1 and IF-2 and the hydrolysis of GTP, the 50S subunit of the ribosome binds to form the 70S initiation complex.

Elongation of Protein Synthesis

• The initiation codon (AUG) is located at the P site and is bonded via codon-anticodon base pairing by fMet-tRNAfMet during the commencement of the first elongation cycle.
• The A location in the mRNA is where the following codon is located.
• The elongation cycle, which consists of the three processes of aminoacyl-tRNA binding, peptide bond synthesis, and translocation, is how polypeptide chains are extended.

Aminoacyl-tRNA binding

• The matching aminoacyl-tRNA for the second codon binds to the A site through codon-anticodon interaction.
• Elongation factor EF-Tu and GTP are needed for the aminoacyl-tRNA to bind, and they interact to form the complex aminoacyl-tRNA/EF-Tu/GTP.
• After binding, the GTP is hydrolyzed, and the EF-Tu, which is now bound to GDP, is released.
• The EF-Tu molecule must first undergo regeneration via a process involving another elongation factor, EF-Ts, before it can catalyze the binding of another charged tRNA to the ribosome.
• The EF-Tu-EF-Ts exchange cycle is the name given to this regeneration.
• First, the GDP is displaced by EF-binding Ts to EF-Tu. GTP then binds to the EF-Tu and pushes out the EF-Ts. Now prepared for another round of elongation, the EF-Tu-GTP.

Peptide bond formation

• Peptidyl transferase facilitates the synthesis of peptide bonds in the second stage.
• During this process, the carboxyl end of the amino acid connected to the tRNA in the P site splits from the tRNA and forms a peptide bond with the amino group of the amino acid attached to the tRNA in the A site.

Translocation

• The elongation factor EF-G (also known as translocase) and GTP complex, or EF-G/GTP, attach to the ribosome in the third phase.
• Now, there are three coordinated motions that together makeup translocation.
• Deacylated tRNA transitions from the P site to the E site.
• The A site dipeptidyl-tRNA shifts to the P site, and
• The ribosome moves three nucleotides along the mRNA (5′ to 3′) to put
• the codon after that at the A site.
• GTP is hydrolyzed into GDP and inorganic phosphate during the translocation processes, and EF-G is then freed and ready to bind more GTP for further elongation.
• The A site is vacant and available to accept the subsequent aminoacyl-tRNA following translocation.
• It is impossible to occupy both the A and E sites simultaneously. So, before the subsequent aminoacyl-tRNA attaches to the A site to begin a fresh round of elongation, the deacylated tRNA is released from the E site.
• The process of elongation proceeds, adding one amino acid to the developing polypeptide’s C-terminal end for each codon that is read, with the peptidyl-tRNA shifting back and forth between the P site and the A site.

Termination of Protein Synthesis

• One of the three termination codons, also known as stop codons, eventually settles at the A site. UAG, UAA, and UGA are these.
• Prokaryotic cells do not have aminoacyl-tRNAs complementary to other codons, in contrast to other codons.
• Cease codons. One of the two release factors (RF-1 or RF-2) binds as a substitute.
• While RF-2 identifies UAA and UGA, RF-1 only detects UAA and UAG. In order to help RF-1 or RF-2 interact with the ribosome, RF-3, a third release factor, is also required. Thus, depending on the precise termination codon at the A site, either RF-1 + RF-3 or RF-2 + RF-3 bind.
• While RF-3/GTP attaches somewhere else on the ribosome, RF-1 (or RF-2) binds at or close to the A site.
• The releasing factors weaken the bond between the polypeptide and tRNA at the P site by prompting the peptidyl transferase activity to convert the polypeptide to a water molecule instead of aminoacyl-tRNA.
• Now, the ribosome discharges the free polypeptide, followed by the free mRNA and free tRNA, and separates into the 30S and 50S subunits in order to restart translation.

References

• David Hames and Nigel Hooper (2005). Biochemistry. Third ed. Taylor & Francis Group: New York.
• Bailey, W. R., Scott, E. G., Finegold, S. M., & Baron, E. J. (1986). Bailey and Scott’s Diagnostic Microbiology. St. Louis: Mosby.
• Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., & Stahl, D. A. (2015). Brock biology of microorganisms (Fourteenth edition.). Boston: Pearson.