Protein translation of Prokaryotes which is copied informations encoded in the prokaryotic DNA to mRNA & then decoded by the ribosome to produce proteins. The process by which proteins are produced with amino acid sequences specified by the sequence of codons in mRNA (messenger RNA) is term as translation.
A triplet of nucleotides in an mRNA that corresponds to a particular amino acid or serves as a translation termination signal is known as a ‘Codon’. The assignment of each of the possible codons to amino acids/ stop signals is called the ‘Genetic Codon’.
5̍-AUG GUG GCG CUG-3̍
There are 20 amino acids that can be present in a polypeptide chain. And there are 4 nucleotides each of the 3 bases in a codon can be one of these 4 nucleotides. So, there are 64 codon combinations. So, more than one codon is assigned for the same amino acid.
Stop codons- UAA, UAG, UGA
Start codon- AUG
Genetic code is universal. Genetic code is known to be highly redundant (degeneracy). That is several different codons is assigned to a same amino acid. In eukaryotes mRNA contains information for amino acids sequences for different. They are known as ‘Polycistronic’.
The genetic code,
- Codons (5′-3′) are presented in mRNA language.
- 42 = 64 different combinations possible.
- 61 of 64 code for all 20 amino acids.
- UAG, UGA and UAA do not code for amino acids, known as terminator or stop codons.
Characteristics of the genetic code
- Usage of the code is remarkably consistent throughout all living organisms.
- Redundancy – That is several different codons is assigned to a same amino acid.
- Specificity – Specific codons always code for same amino acid.
- Universality – Specificity of the code has been conserved from early stages of the evolution.
- Comma less and nonovrlapping – The code may be read from fixed starting point as a continuous sequences of bases, taken 3 by 3.
Components required for protein translation process
- Amino acids – these are building units of proteins.
- Messenger RNA (mRNA) – Specific mRNA act as a template for translation.
- Transfer RNA (tRNA) – At least one specific type of tRNA required per amino acid.
- Peptidyl tranferase – This enzyme catalyses the incorporation of amino acids into building polypeptide chain.
- Amino-acyl tRNA syntheatases – The group of enzymes helps to attachment of amino acids to their corresponding tRNA.
Translation is the mRNA directed synthesis of polypeptide chain. The process requires all the 3 classes of RNA. During the translation process specific tRNA’s pickup specific amino acids, transfer the amino acids to the ribosome locate them in their proper place on the polypeptide chain according to the message written in the mRNA molecule.
In transcription the bases were located according to the base pairing. But there is no connection between amino acids and bases. So, third party (tRNA is needed).
Amino acids do not have any specific affinity for mRNA. It is the tRNA molecule which acts as the adaptor. Each type of tRNA specifically recognize 3 adjacent bases in an mRNA strand and the tRNA molecule is known to be acylated when it is attached to the specific amino acid that correspond to the anticodon. There are no tRNA molecules with anticodons that is complimentary to stop/ nonsense codons. The amino acid which corresponds to the codon of that mRNA that will be paired with the anticodon of the tRNA will be covalently bound to the 3̍-OH group of tRNA. A specific amine acid is attached to the tRNA molecule and the reaction is catalyzed by a set of enzymes known as ‘Aminoacyl tRNA Synthetase’(aa-TS). Each of these enzymes specifically recognized only one amino acid and one class of tRNA.
There are at least 20 AA-TS. When an amino acid is attached to a tRNA molecules it is said to be acylated/ charged.
When you have thousands of nucleotides in mRNA the ribosome must be bound to the correct side to initiate the protein translation. Protein translation is initiated at an initiation codon and mRNA has sequences called ‘TIR’ (Translation Initiation Regions)/RBS (Ribosome Building Sites) that flag the correct 1st codon that should be recognized by the ribosome translation.
The three bases in initiator codon are AUG. AUG corresponds to the amino acids Met. Sometimes GUG also at as the initiator codon, normally coding for Val. Some have UUG codon. Regardless of what amino acids these two-codon called for when they act as initiator codons, they encode for formyl methionine. So, the initiation codon is not necessarily the first sequence in the mRNA.
Shine- Dalgarno Sequence
The codon AUG is not good enough to serve as the Translation Initiation Region (TIR). So, the initiation codon should be accompanied with another sequence that can be used to define a TIR for ribosome.
Shine-Dalgarno Sequence is complimentary to the short sequence near the 3’end of the 16s rRNA. Therefore, it has to properly align the ribosome and the mRNA.
A specially acylated tRNA molecule is required for the translation initiation. It is called ‘Formyl Methionine tRNA’ (FMet-tRNA). The attachment of the formyl group resembles a peptidyl tRNA rather than a normal amino acylated tRNA. For the synthesis of formyl methionine tRNA fMet the amino cycle tRNA synthetase of the normal tRNA Met to the tRNA and then the enzyme transfomylase adds a formyl group to the amino group of methionine.
Generally, to initiate the translation 30s ribosome subunit binds to the S-D Sequence. However, translation is not started until the ribosome reaches the start codon. There are several different initiation factor proteins involve in the translation initiation process. IF3 is the initiation factor that dissociate 30s and 50s subunits by binding into 30s subunit. The next initiation factor is IF1. IF1 binds to the amino acyl site of the 30s subunit.
The initial binding of formyl methionine tRNA fMet does not depend on the initiator codon. However, IF2 adjust the mRNA initiator codon and formyl methionine tRNA and make the binding codon specific. IF2 with the help of GTP (Guanosine-5′-triphosphate), helps to bind the formyl methionine tRNA to the 30s ribosomal subunit.
The initial binding of formyl methionine tRNA fMet does not depend on the initiator codon however IF2 adjust the mRNA initiator codon and formyl methionine tRNA fMet and make the binding codon specific. And the IF1 and IF3 are ejected. The If2 promotes the binding of 50s subunit of the ribosome. In the process IF2 is released with the cleavage of GTP into GDP.
The P site of the 50s subunit in 70s ribosome is occupied by the formyl methionine tRNA fMet. So, this complex is called ‘70s Initiation Complex’.
There are three sites in the ribosome where the tRNA molecules interact with the mRNA.
- Aminoacyl site (A)
- Peptidyl site (P)
- E site (E)
Protein Translation Elongation
The joining of individual amino acids to form a polypeptide chain is known as elongation. During the translation elongation the ribosome moves 3 nucleotides at a time along the mRNA to 5’®3̍ direction. At the beginning the P site of the ribosome is filled with formyl methionine tRNA and the A site can accept any tRNA molecule which has the anti-codon that can pair with a codon located within the A site. The entry of aminoacyl tRNA is random. Amino acylated tRNA (aa tRNA) is delivered to the ribosome by a protein factor called ‘Elongation Factor’-TU(EF-TU). Amino acylated tRNA is complexed with EF-TU and GTP, if the anti-codon is complimentary to the mRNA codon at the acylated site then base pairing occur.
The accurate codon anti-codon pairing can cause conformational changes of the ribosome and this triggers the hydrolysis of GTP on EF-TU-GTP complex then EF-TU-GDP complex is released. When the matching amino acids tRNA is bound at the A site peptide bond formation between the 2 amino acids in the P site and the A site occurs. The reaction is catalyzed by the peptidyl transferase enzyme activity of the 23s rRNA in 50s subunit.
As the peptide bond is formed formyl methionine is cleaved from the formyl methionine tRNA molecule and the uncharged tRNA occupies the P site and the acyl contains the dipeptidyl tRNA. After this 3 movements are occurring.
- The uncharged tRNA leaves the P
- Peptidyl tRNA moves from A site to P
- The ribosome moves a distance of 3 nucleotides to position the next codon at the A This step is known as ‘Translocation’. The protein factor is ‘Elongation factor-G’ (EF-G/ translocase).
In this EF-G is complexed with GTP involves in the translocation of dipeptidyl tRNA and the mRNA. When this movements occur the A, site is again available to accept another amino acid tRNA with the correct anti-codon. This process continues adding amino acids to the growing polypeptide chain. The third tRNA molecule is bound to an adjacent site called the E site (for exit).
The elongation step continues and translation proceeds along the mRNA one codon at a time, until the ribosome encounters of the stop the 3 codons. These stop codons do not have any corresponding tRNA. Termination of translation also requires release factors (RF). These proteins recognize the nonsense codons in the ribosomal A site and promote the release of polypeptide chain from the tRNA and also the ribosome from the mRNA. In E. coli there are 2 release factors.
- RF1-recognizes UAA, UAG
- RF2-recognizes UGA
T.A.BROWN. (2010). GENE CLONING & DNA ANALYSIS (6th ed.). A John Wiley & Sons, Ltd,Publication.
Schleif, R. (2015). Genetics and Molecular Biology (2nd ed.). The Johns Hopkins University Press Baltimore and London.
Pasindu Chamikara – Microbiologist