Protein Synthesis

Course Instructor
Dr. Anoop Kumar Gupta
Department of Chemical and
Biochemical Engineering
Email: anoopg@iitp.ac.in

INDIAN INSTITUTE OF TECHNOLOGY PATNA

1
 Gene Expression

 Gene expression is the transfer of genetic

information (codes) from DNA to RNA to
synthesize proteins in ribosomes.
 Involves two main steps:
1) Transcription (coding step, RNA synthesis
from DNA)
2) Translation (decoding step, Protein synthesis
from RNA)
 Eukaryotes: i) Transcription takes place in
Nucleus.
ii) Translation takes place in
Cytoplasm.
 Prokaryotes: Both Transcription and Translation
take place in Cytoplasm.
2
RNA and its types  RNA is a polymer of nucleotides (5-
carbon ribose sugar + phosphate +
A,G,C,U (not T) nitrogenous bases)
which are held together by 3′-5′
phosphodiester bond.

 It transmits the genetic information

from DNA to make proteins.

 Mainly three types:

1) mRNA (messenger RNA): carries
genetic code copied from DNA during
transcription in form of triplets of
nucleotides called codons, and passes this
information to ribosomes, ~5-10% of
total RNA in cell, made in Nucleoplasm.
3
In DNA In RNA

4
 Amino acid

Different RNA types

2) tRNA (transfer RNA): stem loop structure, brings amino acids to ribosome to make
proteins, each of the 20 amino acids has a specific tRNA that binds with it and
transfers it to the growing polypeptide chain, carries anticodons complimentary to
mRNA codon sequence, 10-15% of total RNA, made in Nucleoplasm.

3) rRNA (ribosomal RNA): main constituent of ribosomes where protein synthesis

occurs ~75% of total RNA, ribosomes are composed of small and large subunits which
have their own rRNA molecules, made in Nucleolus.
5
Structure of tRNA

6
 Genetic codes
 Codon: 3 base sequence (triplet) in RNA in 5′-3′
direction.
 Each codon determines code for an amino acid.
 4 different bases (A,G,C,U), group of 3 bases, 43 = 64
possible combinations.
 64 codons in genetic code vs. 20 amino acids.
 Certain codons signal the “start” and “end” of a
polypeptide chain.
 The codon AUG (represents Methionine amino acid)
is considered as start codon.
 UAA, UAG, UGA are stop codons that signal the end
of protein synthesis (translation)
 Each genetic code correspond only to one amino acid
(non-ambiguous)
 More than one triplet can represent the same amino
acid (degenerative). 7
 Genetic codes (contd.)

 Genetic codes are universal (same for prokaryotes and eukaryotes)

8
 Part 1-Transcription: synthesis of mRNA

 Transcription is a process of creating an equivalent (complimentary and antiparallel)

single-stranded RNA copy from a sequence of DNA.
 During transcription
• A section of DNA (gene) unwinds into two separate strands. 9
 Transcription (contd.)

• One strand of DNA is used as a template strand (3′-5′ direction) while the other
is called coding (or non-template) strand (5′-3′ direction).
• The coding strand has the same sequence as RNA transcript (except for
substitution of U instead of T)
• mRNA is synthesized using complementary base pairing with uracil (U) replacing
thymine (T).
• The enzyme DNA dependent RNA Polymerase moves along the DNA template to
synthesize the corresponding mRNA.
• In Eukaryotes, there are three types of RNA polymerase: RNA Polymerase-I (for
rRNA), RNA Polymerase-II (for mRNA) and RNA Polymerase-III (for tRNA).
• Ribonucleotide monomers required: ATP, GTP, CTP, UTP

 Transcription is highly selective due to signals embedded in nucleotide sequence of

DNA.
10
 Transcription (contd.)
Coding strand Transcription bubble

Template strand

 Specific sequences mark the beginning and end of DNA segment which is to be
transcripted.
 These signals instruct the RNA Polymerase enzyme where and when to start and stop.
11
 Transcription (contd.)
 During transcription, a DNA sequence is read by an RNA Polymerase in 3′-5′
direction, which produces a complementary, antiparallel RNA strand in 5′-3′ direction.
 The template DNA consists of three regions: a promoter, a structural gene
(transcription unit), and a terminator.
 Three steps in transcription:

1) Initiation:
• RNA Polymerase binds to promoter (a specific sequence on coding strand) at 3′-
end of DNA to initiate transcription.
• RNA Polymerase creates a transcription bubble, which separates (unwinds and
unzips) the two strands of the DNA helix. This is done by breaking the hydrogen
bonds between complementary DNA nucleotides.
• No primer is needed to begin transcription.
• Sigma (σ) protein is the initiation factor in RNA Polymerase which identifies the
promoter region on DNA. 12
 Transcription (contd.)

2) Elongation:
• RNA Polymerase adds ribonucleotides (which are complementary to the deoxy-
ribonucleotides of one DNA strand) at 3′-hydroxyl end of growing mRNA chain.
• RNA Polymerase lacks a 3′-5′ exonuclease proofreading activity as in case of
DNA Polymerase in DNA Replication.
3) Termination:
• Rho (ρ) protein is termination factor in RNA Polymerase, it dissociates the RNA
Polymerase from DNA strand by breaking the hydrogen bonds of RNA–DNA
helix, thus freeing the newly synthesized RNA strand.
• The RNA may remain in the nucleus or exits to the ribosome in cytoplasm
through the nuclear pores.
• Some post treatment such as splicing (removal of non-coding regions-introns),
capping, poly adenine tail, etc., is required in Eukaryotes to get mature mRNA.
13
14
15
 Part 2- Translation: Protein synthesis from RNA

Transcription: synthesis of RNA

from DNA

 A sequence of codons (nucleotide triplets) in mRNA yields a specific sequence of

amino acids during synthesis of a polypeptide chain or protein.
 mRNA produced in transcription is decoded in the ribosome to produce a specific
protein according to the rules specified by the genetic code.
 tRNAs carry specific amino acids that are chained together into a polypeptide as
mRNA passes through and is read by the ribosome.
 ATP, GTP molecules act as energy source for various steps involved in Translation.
16
 Translation (contd.)

 Second stage of gene expression which provides pathway for protein synthesis.
 It occurs in Cytoplasm where Ribosomes are located.
 The process begins after mRNA enters into the Cytoplasm through nuclear pores.
 mRNA is used as a template to synthesize chain of amino acids that form a
protein.
 Amino acids are brought to the Ribosomes by tRNA molecules and assembled
into proteins in form of polypeptides.
 Translation is more complex in Eukaryotes than in Prokaryotes.

17
Role of Ribosome in Translation

Translation direction

18
 Steps involved in Translation

1) Activation:
• An amino acid is covalently bonded to tRNA to form aminoacyl tRNA.
• Formation of aminoacyl tRNA occurs in presence of Aminoacyl tRNA
Synthetase enzyme by formation of ester bond between 3′-OH end of tRNA and
carboxyl group of amino acid.
• ATP acts as energy source for the activation of amino acids.
• tRNA carries the amino acid that matches with the triplet codes of mRNA.
• When a tRNA has an amino acid linked to it, it is termed “charged”.
• 20 different enzymes for 20 different amino acids.
Mg+2
Amino acid + tRNA + ATP ⟶ aminoacyl-tRNA + AMP + 2Pi

19
Activation of amino acid

20
 Steps involved in Translation (contd.)

2) Initiation:
• The small and large subunits of Ribosome associate
and mRNA binds between them at 5′ end to form
the initiation complex.
• mRNA molecule is read in 5′-3′ direction as
Ribosome moves along mRNA strand to search for
start AUG codon present on mRNA strand.
• The start codon AUG is read by A site.
• The initiating aminoacyl-tRNA (Methionine tRNA)
having complimentary anticodon (UAC) basepairs
with the AUG codon at P site of the Ribosome.
• A new tRNA with the next amino acid approaches
to A site and a peptide bond forms between the two
adjacent amino acids at the first and second codons.
21
 Initiation step during Translation
• The assembled
Ribosome has one
mRNA binding site
on small subunit and
3 tRNA binding
sites (A, P, E) on
large subunit.
• Aminoacyl tRNA site
(A site: acceptor)
• Peptidyl tRNA site
(P site: donor)
• Exit (E site)

22
 Steps involved in Translation (contd.)

3) Elongation:
• tRNA transfers an amino acid to the polypeptide
corresponding to the next codon.
• The ribosome then moves (translocation) to the next
mRNA codon to continue the process, creating an amino
acid chain on P site of the Ribosome.
• During translocation:
 the ribosome shifts to the adjacent codon in 3′ direction on
the mRNA.
 tRNA on A site moves to P site and peptide bond is
formed with the existing polypeptide at P site.
 The uncharged tRNA at P site is translocated to E site
and leaves the Ribosome.
 a new aminoacyl tRNA binds to the A site.
 This process continues until a stop codon is encountered.
23
Elongation step

24
Translocation

Peptide bond between two amino acids

25
 Steps involved in Translation (contd.)

4) Termination
• When any of three stop codons (UAA, UAG, UGA) appear on A site of
Ribosome, no tRNA can recognize it and therefore no further addition of
amino acid to the growing polypeptide chain.
• Polypeptide chain thus formed is detached and released from E site of
Ribosome.
• Polypeptide chain later folds into proper 3D conformation to give final
form of protein and performs its functions in the cell.
• The two subunits of the Ribosome dissociate.
• The new translation begins.

26
Overall Translation
Process

27
 Summary of Translation

 mRNA strand produced from Transcription attaches to a Ribosome.

 Ribosome identifies the START codon on mRNA strand and translation

initiates.

 tRNA molecules (bonded to specific amino acids) attach to the codons on

mRNA.

 Peptide bonds form between an amino acid and the growing peptide chain.

 The ribosome shifts to each codon on the mRNA in 5′-3′ direction until it reach
the STOP codon.

 The polypeptide chain detaches from the Ribosome to function as an

biologically active protein.
28
Videos to watch
• https://www.youtube.com/watch?v=DKgJPhvCDU8

• https://www.youtube.com/watch?v=QcBYTA7uVXk

• https://www.youtube.com/watch?v=2BwWavExcFI

29