Translation -in prokaryotes-
pooling of charged tRNAs
Prokaryotic Translation (Protein Synthesis)
• Translation involves translating the sequence of a messenger RNA
(mRNA) molecule to a sequence of amino acids during protein
synthesis
• It is the process in which ribosomes in the cytoplasm or ER synthesize
proteins after the process of transcription of DNA to RNA.
Ribosomes
• Ribosomes exist normally as separate subunits that are composed of
protein and rRNA.
• The subunits come together to form a ribosome when they bind to an
mRNA, near its 5’ end.
• On binding to an mRNA, the ribosome reads the nucleotide sequence from
the 5’ to 3’ direction, synthesizing the corresponding protein from amino
acids in an N-terminal (amino-terminal) to C-terminal (carboxyl terminal)
direction.
• Ribosomes are located in the cytosol, either freely floating or associated
with the endoplasmic reticulum.
• They serve to synthesize proteins.
Ribosomal sites for Protein Translation - THREE
1. Aminoacyl-tRNA binding site (or A site) is where, during elongation,
the incoming aminoacyl-tRNA binds.
2. Peptidyl-tRNA binding site (or P site) is where the tRNA linked to the
growing polypeptide chain is bound.
3. Exit site (or E site) is a binding site for tRNA following its role in
translation and prior to its release from the ribosome.
• All three sites (A, P and E) are formed by the rRNA molecules in the ribosome.
Protein synthesis/ translation - Three stages
• During initiation, the mRNA–ribosome complex is formed and the first
codon (always AUG) binds the first aminoacyltRNA (called initiator
tRNA)
• During the elongation phase, the other codons are read sequentially
and the polypeptide grows by addition of amino acids to its C-
terminal end. This process continues until a termination codon (Stop
codon), which does not have a corresponding aminoacyl-tRNA with
which to base pair, is reached.
• At this point, protein synthesis ceases (termination phase) and the
finished polypeptide is released from the ribosome.
Activation of aminoacids
• The activation of aminoacids take place in cytosol.
• The activation of aminoacids is catalyzed by their aminoacyl tRNA
synthetases.
• All the 20 aminoacids are activated and bound to 3’ end of their specific
tRNA in the presence of ATP and Mg++.
• The N-formylated methionine is chain initiating aminoacid in bacteria
whereas methionine is chain initiating aminoacid in eukaryotes.
• Methionine is activated by methionyl-tRNA synthetase. For N-
formylmethionine two types of tRNA are used ie. tRNAmet and tRNAfmet.
• Similarly, all 20 aminoacids are activated (amino acyl-AMP enzyme
complex) and then bound to their specific tRNA forming Aminoacyl tRNA.
Initiation
• In the first step, initiation factor-3 (IF-3) binds to 30S ribosomal unit.
• Then mRNA binds to 30S ribosomal subunit in such a way that AUG codon
lie on the peptidyl (P) site and the second codon lies on aminoacyl (A) site.
• The tRNA carrying formylated methionine ie. FMet–tRNAFMet is placed at
P-site. This specificity is induced by IF-2 with utilization of GTP. The IF-1
prevent binding of FMet–tRNAFMet is in A-site.
• Shine Dalgarno sequence in the mRNA guide correct positioning of AUG
codon at P-site of 30S ribosome.
• After binding of FMet–tRNAFMet on P-site, IF-3, IF-2 and IF-1 are released
so that 50S ribosomal unit bind with 30S forming 70S ribosome. The exit
site is located in 50S.
• The Shine–Dalgarno (SD) sequence is a ribosomal binding site in
bacterial and archaeal messenger RNA, generally located around 8
bases upstream of the start codon AUG.
• The RNA sequence helps recruit the ribosome to the messenger RNA
(mRNA) to initiate protein synthesis by aligning the ribosome with the
start codon.
Elongation of Protein Synthesis
• At the start of the first round of elongation, the initiation codon (AUG)
is positioned in the P site with fMet-tRNAfMet bound to it via
codon–anticodon base pairing.
• The next codon in the mRNA is positioned in the A site.
• Elongation of the polypeptide chain occurs in three steps called the
elongation cycle, namely aminoacyl-tRNA binding, peptide bond
formation and translocation:
Elongation
Binding of AA-tRNA at A-site:
• The 2nd tRNA carrying next aminoacid comes into A-site and
recognizes the codon on mRNA. This binding is facilitated by EF-TU
and utilizes GTP.
• After binding, GTP is hydrolysed and EF-TU-GDP is releasd
• EF=TU-GDP then and enter into EF-TS cycle.
Peptide bond formation:
• The aminoacid present in t-RNA of P-site ie Fmet is transferred to t-
RNA of A-site forming peptide bond. This reaction is catalyzed by
peptidyltransferase.
• Now, the t-RNA at P-site become uncharged
Ribosome translocation:
• After peptide bond formation ribosome moves one codon ahead along 5’-
3’ direction on mRNA, so that dipeptide-tRNA appear on P-site and next
codon appear on A-site.
• The uncharged tRNA exit from ribosome and enter to cytosol.
• The ribosomal translocation requires EF-G-GTP (translocase enzyme) which
change the 3D structure of ribosome and catalyze 5’-3’ movement.
• The codon on A-site is now recognized by other aminoacyl-tRNA as in
previous.
• The dipeptide on P-site is transferred to A-site forming tripeptide.
Termination
• The peptide bond formation and elongation of polypeptide continues
until stop codon appear on A-site.
• If stop codon appear on A-site it is not recognized by t-RNA carrying
aminoacids because stop codon do not have anticodon on mRNA.
• The stop codon are recognized by next protein called release factor
(Rf-1, RF-2 and RF-3) which hydrolyses and cause release of all
component ie 30s, 50S, mRNA and polypeptide separates.
• RF-1 recognizes UAA and UAg while RF-2 recognizes UAA and UGA
while RF-3 dissociate 30S and 50S subunits.
• In case of eukaryotes only one release actor eRF causes dissociation.
