Prokaryotic translation (protein synthesis)
Translation
In molecular biology and genetics the term translation in broadly is the gradual process of synthesis of protein from RNA (tRNA and mRNA) where ribosomes in the cytoplasm are involved and it occurs after the process of transcription of DNA to RNA in the nucleus of the cell completes. This entire process of formation of protein from DNA is called gene expression.
Ribosomal Sites
A prokaryotic ribosome
generally has three different sites for the binding of tRNAs, these are:
§ The aminoacyl-tRNA binding site or aminoacyl site (or A site): This is the site where during elongation the incoming aminoacyl-tRNA binds to.
§ The peptidyl-tRNA
binding site or peptidyl site (or P site): In this site the first tRNA containing
the start codon AUG is linked to the growing polypeptide chain.
§ The exit site (or E site): It is a definite site of ribosome from where the tRNA is released from the ribosome.
Thus a ribosome altogether have three specific sites; Aminoacyl
site (or A site), Peptidyl site(or P site) and Exit site (or E site).
Aminoacylation
§ Amino acid activation (also known as
aminoacylation or tRNA charging) refers to the attachment of an amino acid to
its Transfer RNA (tRNA).
§ This complete process of activation of amino acids
take place in cytosol.
§ The complete process of activation of amino acid
is catalyzed by the enzyme, aminoacyl tRNA synthetase.
§ This activation of tRNA involves two steps:
- Aminoacyl transferase binds Adenosine triphosphate (ATP) to amino acid and forms aa-AMP complex (AMP- amino acid complex) and PPi (pyrophosphate) is released during this process.
- Then the enzyme aminoacyl tRNA
synthetase binds aa-AMP complex to tRNA and the AMP is released in this
process.
The above two processes are commonly called as
coupling reaction which is shown below:
- aa + ATP ⟶ aa-AMP + PP, (pyrophosphate)
- aa-AMP + tRNA ⟶ aa-tRNA + AMP
§ All the 20 amino acids present gets activated by
binding to the 3’ end of their specific tRNA in the presence of ATP and Mg++.
§ N-formylated methionine is the first amino acid
which binds to the tRNA and starts the complete process of translation in
bacteria and the amino acid methionine starts the translation process in
eukaryotes.
§ The amino acid methionine gets activated by an
enzyme – methionyl-tRNA synthetase and for N-formyl methionine two types of
tRNA are required, these are: tRNAmet and tRNAfmet.
§ Similarly in this manner all the 20 amino acids
are also activated (amino acyl-AMP enzyme complex) and then gets bound to their
respective tRNAs and forms amino acyl tRNA.complex.
1. Initiation:
§ It is the first step of the process of translation where the process of protein synthesis starts.
§ initiation factor-3 (IF-3) binds to the 30S sub-unit of the ribosome.
§ Soon then the mRNA binds to the 30S sub-unit of
the ribosome in such a manner that the AUG codon fits on the peptidyl site (or P
site) and the second codon on the aminoacyl site (or A site).
§ The tRNA that carries formylated methionine (i.e., FMet–tRNAFMet ) is placed at the peptidyl site (or P-site).
§ This specificity of binding of tRNA at the
specific site is mainly induced by IF-2 (or initiation factor-2) with the
consumption of GTP (Guanosine Triphosphate).
§ The IF-1 (or initiation factor-1) prevents the
binding of FMet–tRNAFMet complex at the aminoacyl site (or A-site).
§ The shinedalgrno sequence in mRNA guides the correct
and proper positioning of AUG codon at P-site (or peptidyl site) of the 30S
ribosomal subunit.
§ After the binding of FMet–tRNAFMet complex at the P-site (peptidyl site), the initiation factor 3, 2 and 1 (i.e., IF-3, IF-2 and IF-1) are released so that the 50S sub-unit of the ribosome binds to the 30S sub-unit forming 70S ribosome.
2. Elongation:
i. Binding of AA-tRNA at A-site:
§
The second and the next successive tRNAs carrying specific
amino acid gradually comes into A-site of the ribosome and recognizes their
specific codon on mRNA and binds on it.
§
This binding of amino acid-tRNA complex on the codon is
facilitated by EF-TU complex by utilizing GTP.
§ After
binding, the GTP molecule gets hydrolysed and EF-TU-GDP complex is released.
§ The
EF-TU-GDP complex then enters into EF-TS cycle.
ii. Peptide
bond formation:
§ The
amino acid which is present in the tRNA at the P-site of the ribosome (i.e.,
Fmet) is transferred to the tRNA of the A-site forming a peptide bond and this
reaction is catalyzed by an enzyme peptidyltransferase.
§ Then
the tRNA at P-site becomes uncharged.
iii. Ribosome
translocation:
§ After
the formation of peptide bond, ribosome gradually moves one codon ahead along
5’ Ã 3’
direction on mRNA chain.
§ This
is done so that the dipeptide tRNA appears on P-site and the next codon appears
on A-site.
§ The
uncharged tRNA is released out through E-site (or exit site) of ribosome and enters
into the cytosol.
§ The
ribosomal translocation requires EF-G-GTP (translocase enzyme) to change the 3D
structure of ribosome and catalyzes 5’ Ã 3’ movement.
§ On
A-site the codon is then recognized by other aminoacyl-tRNA as earlier.
§ The
dipeptide formed on P-site is then transferred to the A-site of the ribosome
and forms tripeptide.
§ This process of formation of peptide bonds further continues giving rise to a long polypeptide chain of amino acids.
3. Termination:
§ In this step of translation the complete polypeptide thus formed gets separated from the tRNA and functions independently in the cytosol.
§ The process of gradual formation of peptide bond
and elongation of polypeptide chain continues until a stop codon appears on the
A-site of the ribosome.
§ When a stop codon appears on the A-site of the
ribosome then it does not get recognized by any t-RNA carrying aminoacids as
stop codons don’t have anticodon loop on mRNA.
§ The stop codons are then recognized by certain
proteins called release factor (Rf-1, RF-2 and RF-3) which gets hydrolysed and
causes release/separation of all the components which are joined together (i.e.,
30s, 50S, mRNA and polypeptide).
§ The release factor RF-1 recognises the stop
codons UAA and UAG, while the RF-2 release factor recognises the stop codons
UAA and UGA and RF-3 release factor dissociate 30S and 50S ribosomal subunits.
§ In case of eukaryotic translation only one release actor eRF causes all
these dissociation/separation.
Post translation modification:
The long newly formed
polypeptide chain produced at the end of translation may not be biologically
functional. Thus, it undergoes several foldings and processings which is
collectively called as post translation modification. During this post
translational modification the polypeptide undergoes following changes to
become functional protein, these are given below:
1. Amino terminal and carboxyl terminal modification:
In bacteria or in prokaryotes this type of modification occurs by
removing N-formylmethionine
from polypeptide chain and certain carboxyl terminals are also removed by the enzymatic
action in order to make functional protein. While in case of eukaryotic protein
formation the amino terminal of the polypeptide is N- acetylated.
2. Loss of signal sequences:
The amino terminal end of
some protein is cleaved by a specific peptidase enzyme so that the protein loses
its signaling property.
3. Modification of individual amino acids:
In this case the amino acids may get phosphorylated, acetylated for modification.
4. Attachment of carbohydrate side chain:
Side chain of lipid, glucose and many such other group is added to
the polypeptide to make the protein functional. Eg, glycoprotein. Lipoprotein
5. Addition of isoprenyl group:
Isoprenyl group is also added to some proteins in order to make
them active.
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