Protein synthesis Transcription and Translation

PROTEIN SYNTHESIS
Proteins are giant molecules formed by polypeptide chains of hundreds to thousands of amino
acids. These polypeptide chains are formed by about twenty kinds of amino acids. An amino
acid consists of a basic amino group (-NH2) and an acidic carboxyl group (COOH). Different
arrangement of amino acids in a polypeptide chain makes each protein unique.
Proteins are fundamental constituents of protoplasm and building material of the cell. They
take part in the structural and functional organization of the cell. Functional proteins like
enzymes and hormones control the metabolism, biosynthesis, energy production, growth
regulation, sensory and reproductive functions of the cell. Enzymes are catalysts in most of the
biochemical reactions. Even the gene expression is controlled by enzymes. The replication of
DNA and transcription of RNA is controlled by the proteinous enzymes.
Components of Protein Synthesis:
Protein synthesis is governed by the genetic information carried in the genes on DNA of the
chromosomes.
The main components of the protein synthesis are:
1. DNA
2. Three types of RNAs
3. Amino acids 4. Ribosomes
5. Enzymes.
DNA is the master molecule which posseses the genetic information about the sequence of
amino acids in a polypeptide chain. Structure and properties of DNA regulate and control the
synthesis of proteins.
DNA present in the nucleus sends out information in the form of messenger RNA into the
cytoplasm, which is the site of the protein synthesis in eukaryotes. The messenger RNA carries
the information regarding the sequence of amino acids of the polypeptide chain to be
synthesized. This message or information is in the form of a genetic code. This genetic code
specifies the language of amino acids to be assembled in a polypeptide.The genetic code is
deciphered or translated into a sequence of amino acids.
Composition of Genetic Code:
DNA molecule has three components. They are sugar, phosphates and nitrogen bases. Only
nitrogen base sequence varies in different DNA molecules. Thus, the sequence of nitrogen
bases or nucleotides in a DNA segment is the code or language in which the DNA sends out
the message in the form of messenger RNA (mRNA).
The mRNA carries the genetic message (genetic code) in the form of nucleotide sequence. It
has been found that there is colinearity between nucleotide sequence of mRNA and amino acid
sequence of the polypeptide chain synthesized.
The genetic code is the language of nitrogen bases. There are four kinds of nitrogen bases and
twenty kinds of amino acids. Therefore four-letter language of nitrogen bases specifies the
twenty letter language of amino acids.

Mechanisms of Protein Synthesis:
In prokaryotes, the RNA synthesis (transcription) and protein synthesis (translation) take place
in the same compartment as there is no separate nucleus. But in eukaryotes, the RNA synthesis
takes place in the nucleus while the protein synthesis takes place in the cytoplasm. The mRNA
synthesized in the nucleus is exported to cytoplasm through nucleopores.
First, Francis Crick in 1955 suggested and later Zemecnik proved that prior to their
incorporation into polypeptides, the amino acids attach to a special adaptor molecule called
tRNA. This tRNA has a three nucleotide long anticodon which recognizes three nucleotide long
codon on mRNA.
Transcription
Transcription begins with the opening and unwinding of a small portion of the DNA double
helix to expose the bases on each DNA strand. One of the two strands of the DNA double helix
then acts as a template for the synthesis of RNA. Ribonucleotides are added, one by one, to the
growing RNA chain, and as in DNA replication, the nucleotide sequence of the RNA chain is
determined by complementary base-pairing with the DNA template. When a good match is
made, the incoming ribonucleotide is covalently linked to the growing RNA chain in an
enzymatically catalyzed reaction. The RNA chain produced by transcription—the transcript—
is therefore elongated one nucleotide at a time and has a nucleotide sequence exactly
complementary to the strand of DNA used as the template.
Unlike a newly formed DNA strand, the RNA strand does not remain hydrogenbonded to the DNA template strand. Instead, just behind the region where the ribonucleotides
are being added, the RNA chain is displaced and the DNA helix re-forms. For this reason—
and because only one strand of the DNA molecule is transcribed—RNA molecules are singlestranded. Further, as RNAs are copied from only a limited region of DNA, these molecules are
much shorter than DNA molecules. The enzymes that carry out transcription are called RNA
polymerases.
Before a eukaryotic RNA exits the nucleus, it must go through several different RNA
processing steps- capping and polyadenylation.
1. RNA capping involves a modification of the 5’ end of the mRNA transcript, the end that
is synthesized first during transcription. The RNA is capped by the addition of an atypical
nucleotide—a guanine (G) nucleotide with a methyl group attached.
2. Polyadenylation provides newly transcribed mRNAs with a special structure at their
3’ ends. In contrast with bacteria, where the 3’ end of an mRNA is simply the end of
the chain synthesized by the RNA polymerase, the 3’ ends of eucaryotic RNAs are first
trimmed by an enzyme that cuts the RNA chain at a particular sequence of nucleotides
and are then finished off by a second enzyme that adds a series of repeated adenine (A)
nucleotides to the cut end. This poly-A tail is generally a few hundred nucleotides long.
These two modifications—capping and polyadenylation—are thought to increase the stability
of the eukaryotic mRNA molecule, to aid its export from the nucleus to the cytoplasm, and
generally to identify the RNA molecule as an mRNA. They are also used by the proteinsynthesis machinery to make sure that both ends of the mRNA are present and that the message
is therefore complete before protein synthesis begins.

Role of Ribosomes in Protein synthesis:
Ribosome is a macromolecular structure that directs the synthesis of proteins. A ribosome is a
multicomponent, compact, ribonucleoprotein particle which contains rRNA, many proteins and
enzymes needed for protein synthesis. Ribosome brings together a single mRNA molecule and
tRNAs charged with amino acids in a proper orientation so that the base sequence of mRNA
molecule is translated into amino acid sequence of polypeptides.
Small subunit of ribosome contains the decoding centre in which charged tRNAs decode o the
codons of mRNA. Large subunit contains peptidyl transferase centre, which forms the peptide
bonds between successive amino acids of the newly synthesized peptide chain. The mRNA
binds to the 16S rRNA of smaller subunit. Near its 5′-end mRNA binds to the 3′-end of 16S
rRNA.
The main role of ribosome is the formation of peptide bond between successive amino acids
of the newly synthesized polypeptide chain. The ribosome has two channels in it. The linear
mRNA enters and escapes through one channel, which has the decoding centre. This channel
is accessible to the charged tRNAs. The newly synthesized polypeptide chain escapes through
the other channel.
Direction of Translation:
Each protein molecule has an -NH2 end and -COOH end. Synthesis begins at amino end and
ends at carboxyl end. The mRNA is translated in 5′ → 3′ direction from amino to carboxyl end.
Synthesis of mRNA from DNA transcription also occurs in 5′ → 3′ direction.

Initiation of Protein Synthesis:
Formation of Initiation Complex:
First of all 30S subunit of the 70S ribosome starts initiation process. The 30S subunit, mRNA
and charged tRNA combine to form pre-initation complex. Formation of preinitiation complex
involves three initiation factors IF1, IF2 and IF3 along with GTP (guanosine triphosphate).
Later 50S subunit of ribosome joins 30S subunit to form 70S initiation complex.
Information for protein synthesis is present in the form of three nucleotide codons on mRNA.
Protein coding regions on mRNA consist of continuous, non-overlapping triplet codons. The
protein coding region on mRNA is called open reading frame which has a start codon 5′-AUG3′ and a stop codon in the end. Each open reading frame specifies a single protein. Prokaryote
mRNA has many open reading frames, therefore encode multiple polypeptides and are called
polycistronic mRNAs.

Near the 5′-end of mRNA lies the start codon which is mostly 5′-AUG-3′ (rarely GUG) in both
prokaryotes and eukaryotes. Ribosome binding site (RBS) in prokaryotes lies near the 5′- end
of mRNA ahead (upstream) of AUG codon.
Between 5′-end and AUG codon there is a sequence of 20-30 bases. Of these, there is a
sequence 5′-AGGAGGU-3′. This purine rich sequence is called Shine-Dalgarno sequence and
lies 4-7 bases ahead (upstream) of AUG codon.
The 3′-end region of 16S rRNA is 30S subunit has a complementary sequence
3′AUUCCUCCA-5′. This sequence forms base pairs with Shine-Dalgarno sequence for binding
of mRNA to ribosome. Shine-Dalgarno sequence is the ribosome binding site (RBS). It
positions the ribosome correctly with respect to the start codon.

There are two tRNA binding sites on ribosome covering 30S and 50S subunits. The first site
is called “P” site or peptidyl site. The second site is called “A” site or aminoacyl site. Only the
initiator tRNA enters the “P” site. All other tRNAs enter the “A” site.

For every amino acid, there is a separate tRNA. The identity of a tRNA is indicated by
superscript, such as tRNAArg (specific for amino acid Arginine). When this tRNA is charged
with amino acid Arginine, it is written as Arginine-tRNAArg or Arg-tRNAArg. Charged tRNA
is called aminoacylated tRNA.
In bacteria, the first amino acid starting the protein is always formyl methionine (fMet). When
AUG appears as the start codon on mRNA only fMet is incorporated. The tRNA molecule
carrying formyl methionine is called tRNA™61. Therefore the first initiator charged aminoacyl
tRNA is always fMet-tRNAfMet. When AUG codon is encountered in the internal location (other
than the start codon), methionine is not formylated and tRNA carrying this methinine is
tRNAMet.
First of all the charged initiator tRNA called tMet-tRNAfMet occupies the “P” site on ribosome.
This position brings its anticodon and start codon AUG of mRNA together in such a way that
the anticodon of charged tRNA and codon of mRNA form base pair with each other. Thus
reading or translation of mRNA begins.
The “A” site is available to the second incoming charged tRNA whose anticodon forms base
pairs with the second codon on mRNA.
Charging of tRNA:
Attachment of amino acids to tRNAs is called charging of tRNA. All tRNAs at their 3′terminus have a sequence 5′-CCA-3′. At this site amino acids bind with the help of enzyme
aminoacyl tRNA synthases. Charging of tRNA occurs in two steps.
1. Activation of amino acids:
Energy molecule ATP activates the amino acids. This step is catalysed by specific activating
enzymes called aminoacyl tRNA synthatases. Every amino acid has a separate enzyme AARNA synthatases enzyme.
2. Transfer of amino acids to tRNA:
AA-AMP enzyme complex reacts with a specific tRNA and transfers the amino acid to tRNA,
as a result of which AMP and enzyme are set free.

This first AA-tRNA is fMet-tRNAfmet which is amino acid formyl methionine bound to tRNA.
This fixes itself to “P” site on ribosome. After this the second AA-tRNA attaches itself to “A”
site on ribosome. In this way polypeptide chain elongation begins.

Polypeptide Chain Elongation:
Polypeptide chain elongation requires some elongation factors. These elongation factors are
Tu and G.
EF-Tu forms a complex with AA2-tRNA and GTP and brings it to the “A” site of ribosome.
Once the AA2-tRNA is in place at “A” site, the GTP is hydrolysed to GDP and EF- Tu is
released from the ribosome. EF-Tu-GTP complex is regenerated with the help of another factor
Ts.
Formation of Peptide Bond:
The main role of ribosome is to catalyse the formation of peptide bonds between successive
amino acids. In this way amino acids are incorporated into protein.
Now both “P” site and “A” site on ribosome are occupied by charged tRNAs having amino
acids. Peptide bond is formed between two successive amino acids at “A” site. It involves
cleavage of bond between f-Met and tRNA. This is catalysed by the enzyme tRNA deacylase.
Peptide bond is formed between the free carboxyl group (-COOH) of the first amino acid and
the free amino group (- NH2) of the second amino acid at the “A” site. The enzyme involved in
this reaction is peptidyl transferase. After the formation of peptide bond, between two amino
acids, the tRNA at “P” site becomes uncharged or deacylated and tRNA at “A” site now carries
a protein chain having two amino acids. This occurs in 50S subunit of ribosome.

Translocation:
The peptidyl tRNA carrying two amino acids present at “A” site is now translocated to “P”
site. This movement is called translocation. Elongation factor called EF-G control
translocation. This factor G is called translocase. Hydrolysis of GTP provides energy for
translocation and release of deacylated tRNA (free of amino acid).
Translocation also involves movement of ribosome along mRNA towards its 3′-end by a
distance of one codon from first to second codon. This movement shifts the dipeptidyl tRNA
(carrying two amino acids) from “A” to “P” site.
In addition to these two sites P and A, a third site “E” (exit site) on 50 S ribosome is present.
Deacylated tRNA (deprived of amino acid) moves for “P” site to “E” site from where it is
ejected out.
Then the third amino acid (next amino acid) charged on tRNA comes to lie in now empty site
“A”. Then dipeptidyl chain having two amino acids present on P site form peptide bond with
the third amino acid at “A” site. Then the three amino acid chain is translocated to “P” site.
Now the polypeptide chain has three amino acids. This elongation process goes on and on. At
each step a new amino acid is added to the polypeptide chain. After each elongation, ribosome
moves by one codon in 5′ → 3′ direction.
Chain Termination:
The presence of termination codons or stop codons on mRNA causes the polypeptide chain to
be terminated. Synthesis stops when elongation chain comes across stop codons on “A” site.
The stop codons are UAA, UGA and UAG. There is no tRNA which can bind these codons.

There are three release factors in prokaryotes, which help in chain termination. They are RF1,
RF2 and RF3.
Polyribosome or Polysome:
A single mRNA molecule can be read simultaneously by several ribosomes. A polyribosome
or polysome consists of several ribosomes attached to the same RNA. The number of ribosomes
in a polysome depends upon the length of mRNA.

A fully active mRNA has one ribosome after every 80 nucleotides. There may be about 50
ribosomes in a polycistronic mRNA of prokaryotes. Ribosomes move along mRNA in 5′ 3′
direction. There is a gradual increase in the size of polypeptide chain as the ribosomes move
along mRNA towards its 3′-end. Polypeptide chain starts near the 5′-end and is completed near
the 3′-end.

The ribosomes closest to the 5′-end of mRNA have the smallest polypeptide chain, while
ribosomes nearest to the 3′-end have longest chain. Polysome increases the rate of protein
synthesis tremendously. In bacteria protein is synthesized at the rate of about 20 amino acids
per second.
Simultaneous Transcription and Translation in Prokaryotes:
In prokaryotes, all components of transcription and translation are present in the same
compartment. The mRNA molecule is synthesized in 5′ → 3′ direction and protein synthesis
also occurs in 5′ → 3′ direction. In this way mRNA molecule while still under synthesis has a
free 5′-end whose other end is still under synthesis.
Ribosomes bind at free 5′-end and start protein synthesis. In this way the free end (5′end) of
mRNA starts the process of protein synthesis while still attached to DNA. This is called
Coupled Transcription and Translation. This increases the speed of protein synthesis. After the
protein synthesis is completed, the degradation of mRNA molecule by nucleases also starts at
5′-end and proceeds in 5′ → 3′ direction.

Protein Synthesis in Eukaryotes:
Protein synthesis in eukaryotes is basically similar to that of prokaryotes except some
differences.

The ribosomes in eukaryotes are of 80S having 40S and 60S subunits. In eukaryotes the
initiating amino acid is methionine and not f-methionine as in the case of prokaryotes. A special
tRNA binds methionine to start codon AUG. This tRNA is called tRNAi Met. This is distinct
from tRNAMet which binds amino acid methionine to any other internal position in the
polypeptide.
There is no Shine-Dalgarno sequence in eukaryotic mRNA to function as ribosome binding
site. Between 5′-end and AUG codon of mRNA there is a sequence of bases called cap. Small
subunit of ribosome scans the mRNA in 5′ → 3′ direction until it comes across 5′- AUG-3′
codon. This process is called scanning. Initiation factors also closely associated with 3′-end of
mRNA through its poly-A tail. Initiation factors circularize mRNA by its poly-A tail. In this
way poly-A tail also contributes to the translation of mRNA. Eukaryotic mRNAs are
monoisotopic and encode a single polypeptide, therefore have a single open reading frame.
There are ten initiation factors in eukaryotes. They are elF (eukaryotic intiation factors) are
elFI, eIF2, eIF3, eIF4A, eIF4B, eIF4C, eIF4D, eIF4F, eIF5, eIF6.
There are two elongation factors in eukaryotes like prokaryotes. They are eEFl (similar to EFTu) and eEF2 (similar to EF-G).
Eukaryotes have only one release factor eRF which requires GTP termination of protein
synthesis. It recognizes all the three stop codons.
In eukaryotes the mRNA is synthesized in the nucleus, then processed, modified and passed
on into the cytoplasm through nucleopores. The protein synthesis takes place in the cytoplasm.
The mRNA in prokaryotes is very unstable and its life span is of a few minutes only. The
mRNA of eukaryotes is quite stable and has a longer life span extending upto several days.

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