Translation

The process of Synthesis of protein from mRNA with the help of ribosomes is known as
Translation. It takes place in Cytoplasm.
I. Ribosomes: These are the site of protein synthesis. Ribosomes are made from complexes of
RNAs and proteins. The number of ribosomes in a cell depends on the activity of the cell.
Ribosomes are freely suspended in the cytoplasm or attached to the endoplasmic reticulum
forming the rough endoplasmic reticulum.

They exist in two sizes: 70S are found in all Prokaryotes and are free-floating. While 80S found
in all eukaryotic cells – attached to the rough ER (they are rather larger). Here "S" stands for
Svedberg coefficient or unit. The ribosomes are made up of two subunits - a small and a large
subunit. The small subunit reads the mRNA while the large subunit joins the amino acids to form
a chain of polypeptides. Ribosomal subunits are made of one or more rRNA (ribosomal RNA)
molecules and various proteins. The ribosomes and associated molecules are also known as the
translational apparatus.
A site- Site for entry of new tRNA
P site- P site has tRNA containing growing polypeptide chain
E site- through which a tRNA molecules after delivery of AA exits the ribosomes
(II) Ribosomal RNA (rRNA): The ribosomes are the factories of protein synthesis. The
eukaryotic ribosomes are composed of two major nucleoprotein complexes- 60S subunit and 40S
subunit. The 60S subunit contains around 35 different proteins and possesses three different
rRNA namely; 28S rRNA, 5S rRNA and 5.8S rRNA while the 40S subunit contain 50 proteins
and possesses only one type of rRNA i.e., 18S rRNA.
Prokaryotic ribosomes are also made up of two subunits 30S and 50S. 30S subunit has 16S r-
RNA and 50S subunit contains 23S and 5S r-RNA.
(III) Messenger RNA (m-RNA): Messenger RNA is a linear molecule formed inside the
nucleus by the process of transcription from DNA. The sequence of mRNA is complementary to
the template DNA strand. The nitrogenous bases on mRNA strand are arranged in form of
codons, made up of three nitrogenous bases. Messenger RNA carries genetic information
from nucleus (chromosomal DNA) to cytoplasm for protein synthesis. After being
transported to cytoplasm m-RNA combines with ribosomes to form polyribosomes or
polysomes. Each polysome contains several ribosomes (normally five) which form a complex
with m-RNA.
Messenger RNA has been classified as monocistronic and polycistronic. Monocistronic m-RNA
is a type of m-RNA which codes for only one protein whereas polycistronic m-RNA can
synthesis more than one type of protein from same m-RNA molecules. This is because
monocistronic m-RNA contains codons of single cistron whereas polycistronic m-RNA contains
codons for more than one cistron. Monocistronic m-RNA is characteristic feature of eukaryotes
while prokaryotes contain polycistronic m-RNA.

Translation unit consists of mRNA (5’-3’) having start codon (AUG), Region coding for a
polypeptide, stop codon (UAA, UAG, UGA) and untranslated regions (UTRs).

Comparison of the structure of prokaryotic and eukaryotic mRNA

Eukaryotic mRNA is mostly monocistronic. It has a 5’ cap, which is recognized by small
ribosomal subunit. Protein synthesis, therefore, begins at an initiation codon near the 5’ end of
the mRNA. Upstream of the initiation codon contains a non-translatable sequence called 5’ UTR
(5‘-untranslated region) or leader sequence. Similarly, non-translatable sequence at 3’ end after
stop codon is termed as 3’ UTR (3’-untranslated region) or trailer sequence, which varies in
length and sequence.
In prokaryotes, most of the mRNA is polycistronic. In contrast to eukaryotic mRNA, the 5’ end
has no cap-like structure, and there are multiple ribosome-binding sites (called shine-Dalgarno
sequence) within the polycistronic mRNA chain, each resulting in the synthesis of different
protein. Just like eukaryotic mRNA, prokaryotic mRNA also contains 5’ UTR and 3’ UTR. All
mRNA (monocistronic and polycistronic) contains two types of regions- the coding region
(which starts with initiation codon and ends with a stop codon) and untranslated region (5’- and
3’-UTR). A polycistronic mRNA also contain intercistronic regions.

(3) Transfer RNA (t-RNA)

Transfer RNA molecule consists of a single polynucleotide chain folded to form 5 arms. These
five arms are named as acceptor arm, D-arm, anticodon arm, TΨC arm and Variable (extra) arm.
Transfer RNA contains mainly four arms, each arm with a base paired stem. Except acceptor
arm, other arms have a stem and loop it means that this arm has two segments one called as stem
in which complementary base pairing occurs and another part as a loop in which there is base
pairing. As it has already been stated that acceptor arm has only stem and no loop. Beside this
variable arm may or may not have a stem.
1. The acceptor arm: This arm is capped with a sequence CCA (5′ to 3′). The amino acid is
attached to the acceptor arm.
2. The anticodon arm: This arm, with the three specific nucleotide bases (anticodon), is
responsible for the recognition of triplet codon of mRNA. The codon and anticodon are
complementary to each other.
3. The D arm: It is so named due to the presence of dihydrouridine.
4. The TΨC arm: This arm contains a sequence of T, pseudouridine (represented by psi, Ψ) and
C.
5. The variable arm: This arm is the most variable in tRNA. Based on this variability, tRNAs
are classified into 2 categories:
(a) Class I tRNAs: The most predominant (about 75%) form with 3-5 base pairs length.
(b) Class II tRNAs: They contain 13-20 base pair long arm.

Translation
Process of Translation
The process of protein synthesis translates the codons (nucleotide triplets) of the messenger RNA
(mRNA) into the 20-symbol code of amino acids that build the polypeptide chain of the proteins.
The process of mRNA translation begins from its 5′-end towards its 3′-end as the polypeptide
chain is synthesized from its amino-terminal (N-end) to its carboxyl-terminal (C-end). There are
almost no significant differences in the protein synthesis steps in prokaryotes and eukaryotes,
however there is one major distinction between the structure of the mRNAs – prokaryotes often
have several coding regions (polycistronic mRNA), while the eukaryotic mRNA has only one
coding region (monocistronic mRNA). Proteins are synthesized by ribosomes on mRNA strand.
The process of protein synthesis occurs in following steps

1. Initiation
a. Activation of amino acids
b. Transfer of amino acid to tRNA
2. Elongation of polypeptide chain
3. Chain termination

a. Activation of amino acids

Amino acids found to be present in cytoplasm are present in inactive or dormant state. They
cannot undergo the process of protein synthesis in this inactive state. Hence, the process
synthesis begins with activation of amino acids. Each amino acid is catalyzed by enzyme, known
as aminoacyl tRNA Synthetase which results in formation of aminoacyl adenylate-enzyme
complex. Energy in the form of ATP is required for the process. In the presence of ATP, an
amino acid combines with its specific aminoacyl-tRNA synthetase, and Mg 2+ is also required.
These enzymes first bind and hydrolyze ATP to catalyze a high-energy bond between a carboxyl
group of amino acid and α- phosphate of ATP i.e., adenosine monophosphate (AMP).
The other two phosphates (β and γ) molecules of ATP are removed as inorganic pyrophosphates
(PPi) in this reaction. Hydrolysis of pyrophosphate with the help of enzyme pyrophosphatase
provides energy for driving the initial reactions. The activated amino acid is then transferred to
the tRNA, and AMP is released. The energy made available to amino acid during its activation is
later used in formation of peptide bonds.
b. Transfer of amino acid to tRNA

Once the amino acids get activated the next step in protein synthesis is transfer of activated
amino acids to charging of tRNA. It is also known as charging of tRNA. There are about 100
different types of tRNA found in cytoplasm. Each activated amino acid is transferred to its
specific tRNA by formation of an ester bond between carboxylic group (-COOH) of amino acid
and the –OH group attached to either the 2’ or 3’ carbon on the sugar of the terminal adenine
nucleotide of tRNA.

The enzymes synthetase play a key role in activation as well as transfer of activated amino acid.
The enzymes have two active sites. One site is utilized for identifying specific amino acid and
other to recognize specific tRNA molecule. The overall function of enzymes is to bring amino
acid and its specific tRNA molecule together.

Aminoacyl tRNA Synthetases

It is the enzyme that helps in combining amino acid to its particular tRNA. The enzyme is
specific for each amino acid. It is also called aa-activating enzyme. Through the process of
tRNA ‘charging’ each tRNA molecule is linked to its correct amino acid by a group of enzymes
called aminoacyl tRNA synthetases. When an amino acid is covalently linked to a tRNA, the
resulting complex is known as aminoacyl-tRNA.
These enzymes first bind and hydrolyze ATP to catalyze the formation of a covalent bond
between an amino acid and adenosine monophosphate (AMP); a pyrophosphate molecule is
expelled in this reaction. This is called activating the amino acid. The same enzyme then
catalyzes the attachment of the activated amino acid to the tRNA and the simultaneous release of
AMP. After the correct amino acid covalently attached to the tRNA, it is released by the enzyme.
The tRNA is said to be charged with its cognate amino acid.

Initiation Factor
For initiation of protein synthesis different initiation factors (IF) are required. In Prokaryotes
there are three known initiation factors namely IF-1, IF-2, and IF-3. IF-1 and IF-2 are required
for binding of initiation tRNA to 30S ribosomal subunit. Beside this IF-2 is also needed for
binding of guanosine triphosphate (GTP). IF-3 is the initiation factor which is rich in lysine and
arginine residues and helps in binding of 30S ribosomes subunit to mRNA stand all these factors
are found to be located in 30S subunit of ribosome. Eukaryotic translation initiation factors
include eIF1, eIF2, eIF3, eIF4, and eIF5. The eIF4E subunit of eIF4 binds to the 5′ cap structure
on eukaryotic mRNAs.

The synthesis of all proteins starts with the same amino acid, methionine. In E.coli. and in other
eubacteria, the first amino acid in any newly synthesized polypeptide is N-formylmethionine
because the starting methionine amino acid carries a formyl group.

The next step in the process of protein synthesis is formation of 30S initiation complex. mRNA,
30S subunit of ribosome, initiator tRNA containing attached N- formylmethionine, GTP and
initiation factors (IFa-1, IF-2 and IF-3) combine to form a complex called as 30S initiation
complex. The mRNA gets attached to 30S ribosomal subunit to form mRNA -30S complex. IF-3
is required for this process to occur. Besides its role in binding of mRNA with 30S ribosomal
subunit, IF-3 has other functions to such as it affects the conformation of 30S subunit and also it
presents re-association of 30S and 50S subunits of ribosome. Once they are dissociated. Now, to
the above formed mRNA-30S complex, fmet–tRNA is added. IF-2 and IF-1 along with GTP are
required for this process to occur. Among these IF-2 is the most crucial Factor required for
binding of fmet-tRNA to 30S complex.

Now as we have seen that AUG codon present in mRNA works as initiation codon. A specific
nucleotide sequence is present near initiator AUG codon, or the conformation of tRNA is such
that most of the AUG codons are presented to act as initiator codon. After tRNA gets attached to
mRNA-30S subunit complex, 50S subunit of ribosome is added, which contains two binding
sites (A and P) in which two tRNA molecules can bind. A site is called Aminoacyl or Acceptor
site and P site is called Peptidyl or Polymerization site or Donor site. The initiator tRNA, fmet-
tRNA always directly binds to P site. However, all other tRNA (other than initiator tRNA), first
binds to A site and then they are translocated to P site. During attachment of 50S subunit, GTP is
converted to GDP + Pi and initiation factors IF-1 and IF-2 are released for recycling.

Elongation of polypeptide chain

Translation elongation is second step in protein synthesis. During the elongation step the
polypeptide chain adds amino acids to the carboxyl end. The protein chain grows as the ribosome
moves from the 5' -end to the 3'-end of the mRNA. Factors for the elongation of polypeptide
chain are called as elongation factors (EF). In prokaryotes, the delivery of the aminoacyl-tRNA
to ribosomal A-site is facilitated by elongation factors EF-Tu-GTP and EF-Ts, and requires GTP
hydrolysis. EF-Ts (in prokaryotes) function as nucleotide exchange factors and EF-G bring about
translocation of aminoacyl–tRNA from “A” site to “P” site. Elongation factors EF-T4 and EF-T5
in prokaryotes are required for binding of amino acyl tRNA ribosome. EF-T4 also forms a
complex with aa-tRNA and GTP.
Now the initiator tRNA containing initiation amino acid binds to P-site and another tRNA with
second amino acid binds to A-site. Now a peptide bond will be formed between initiation amino
acid (at P-site) and second amino acid (at A-site). The Peptidyl-transferase (found in ribosomal
unit) is an important enzyme which catalyzes the formation of the peptide bonds. The enzymatic
activity is found to be intrinsic to the 23S rRNA found in the large ribosomal subunit. Because
this rRNA catalyzes the polypeptide bound formation reaction, it is named as a ribozyme.

After peptide bond formation, the ribosome moves 3 nucleotides toward the 3'-end of the mRNA
by which tRNA molecule of fmet–tRNA is released from P site. This process is known as
translocation - in prokaryotes, it requires the participation of EF-G-GTP and GTP hydrolysis.
During the translocation, the uncharged tRNA moves from the P to the E site and Peptidyl-tRNA
(to which the growing polypeptide chain is attached) leaves the A site and go to the P site and a
third tRNA comes into A-site. Now a second peptide bond will be formed between second amino
acid and third amino acid. Again, second tRNA from P-site is released and third tRNA from A-
site will be translocated to P-site and A-site will become free for entry of next tRNA molecule.
By this process large number of amino acids will be linked to one another by formation of
peptide bond leading to formation of polypeptide (protein) chain. This is an iterative process that
is repeated until the ribosome reaches the termination codon.
Chain termination

Termination happens when the A site of the ribosome reaches one of the three termination or
stop codons (UAA, UAG or UGA). The process of termination of protein synthesis can be
divided into three parts starting with recognition of termination signal (Stop codons). Followed
by release of polypeptide chain and finally processing of polypeptide chain. Elongation of
polypeptide chain continuous until a stop codon is read on mRNA. As we already know that
these three codons are regarded as non-coding since they do not code for any amino acid, hence
when the moving ribosome will reach a point where any of these codons is present the process of
protein synthesis will terminate.

Since the synthesis of polypeptide chain stops, the polypeptide chain still remains attached to
tRNA molecule. There are several release factors which brings about hydrolysis of polypeptide
chain at P-site. As a result of action of release factors tRNA is released from P site, ribosome
dissociates into 30S and 50S subunit in prokaryotes. In prokaryotes there are three known release
factors RF1, RF2 and RF3. Release factors RF1 and RF2 recognize stop codons which ultimately
results in termination of protein synthesis. RF3 does not recognize any stop codon but it helps in
stimulating binding and release of RF1 and RF2 from the ribosome. For this reason RF3 is also
called as stimulatory factor or S factor.