300L Medical/Dental Students

By Taiwo T. Adeniyi

1
 Protein Synthesis
 The production or synthesis of polypeptide
chains (proteins)
 Two phases:
Transcription & Translation
Replication at a glance

SSBs DNA
Polymerase III
Helicase
Leading Strand

Lagging DNA Ligase

Primase Strand Polymerase I
DNA Pol
Primer
III
 The information available in the DNA is passed to
mRNA(Transcription) which is then used for
synthesis of a particular protein(Translation).
This is the central dogma of molecular biology.

 Translation takes place with the help of

Ribosomes. Transfer RNA(tRNA), ribosomal
RNA(rRNA), messenger RNA(mRNA), protein
factors and enzymes interact with the ribosomal
protein synthesizing machinery to make a
particular protein.
 DNA  RNA  Protein
Nuclear
membrane
DNA
mRNA must
be
processed
Transcription
Pre-mRNA

before it RNA Processing

leaves the mRNA
nucleus of
Ribosome
eukaryotic
cells Translation

Protein
 Transcription
- The process of copying the sequence of
one strand of DNA, the template strand to
produce mRNA copy
-It requires the enzyme RNA Polymerase
-It occurs in the 5’ to 3’ direction
6
 After the DNA is transcribed into RNA, editing must be
done to the nucleotide chain to make the RNA
functional

 Introns, non-functional segments of DNA are snipped

out of the chain
• Exons, segments of DNA that code for proteins, are then
rejoined by the enzyme ligase

• A guanine triphosphate cap is added to the 5” end of

the newly copied mRNA

• A poly A tail is added to the 3’ end of the RNA

• The newly processed mRNA can then leave the nucleus

Result of Transcription is processed

CAP New Transcript Tail

 Transfer RNA (tRNA): They transfer amino acids
from the cytoplasm to the ribosomal protein
synthesizing machinery

 They are very soluble and present in the

cytoplasm. They are much shorter than the mRNA
(73-93 nt long)

 They show extensive internal base pairing and

acquire a clover leaf-like structure.

 They contain a significant proportion of unusual

bases. E.g. Dihydrouracil(DHU), pseudouridine,
hypoxanthine and many methylated bases.
 Acceptor arm: (3’ end) It
carries the amino acid. The
end sequence is CCA-3’ and
the OH group at this end
forms an ester bond with
COOH end of amino acid.

Anticodon arm: It has the

anticodon that recognises
the triplet nucleotide codon
present in mRNA. The
specificity of tRNA resides in
Hydrogen the anticodon site which has
bonds base sequence
Anticodon complementary to that of
mRNA. E.g. codon= UUU;
tRNA structure anticodon= AAA, aa= Phe
 The tRNA act as adaptor molecules between
mRNA and the amino acids coded by it. i.e. tRNA
is the mediator between mRNA and amino acids

 The D arm or DHU region: It serves as the

recognition site for the enzyme which adds the
amino acid

 The pseudouridine arm (psi arm): It is involved in

binding tRNA to ribosomes.

 About 75% of tRNA have a short extra arm, about

3-5 bp long(class 1) or 13-21 bp long(class 2)
 Ribosomal assembly is the protein synthesizing
machinery. Ribosomes provide necessary infrastructure
for the mRNA, tRNA and amino acids to interact with
each other for the translation process

 -each has three binding sites for tRNA on its surface(P,

A, E)
-each has one binding site for mRNA
-facilitates codon and anticodon bonding
-components of ribosomes are made in the nucleus and
exported to the cytoplasm where they join to form one
functional unit.

 Mammalian ribosome has a sedimemtary velocity of 80S

unit. It has larger 60S subunit and a smaller 40S subunit
 They contain different rRNAs and specific proteins. rRNA
has catalytic activity. (ribozyme)
 Large subunit
Exit Peptidyl-tRNA binding site
site
Aminoacyl-tRNA binding site
E
5’ P A
3’
mRNA

Small subunit
 The messenger RNA contain the coded message that is
translated into protein. The sequence of nucleotides on the
mRNA is read in triplet of bases called codon

 The 4 different bases (A, G, C, U) generate 64 codons or code

words (by permutations and combinations). This is the genetic
code. E.g. UUU=Phe, UCU=Ser, AUG=Met etc

 There are 31 tRNA species, carrying 20 aa to translate 61 codons

(3 codons do not code for any particular amino acids). They are
nonsense or stop codons.

 The mRNA is polyadenylated at its 3’ end. The poly-A tail may be

20-250 nt long. This may be to protect the mRNA against
exonuclease activity

 Eukaryotic mRNA has a molecular ‘cap’ at the 5’ terminus. This is

7-methylguanosine triphosphate. It is useful in recognition of
mRNA by the translating machinery
 The process of translation requires a genetic code
through which the information contained in mRNA
sequence is expressed to produce a specific
sequence of amino acids

 The letters AGCU correspond to nucleotides in RNA

that are organised into triplet of nucleotides called
codons

 The collection of codons is called Genetic code

 Genetic code is like a dictionary that correspond

sequence of nucleotides with sequence of amino
acids

 There are 64 codons in total, 3 of which are non-

sense codons(stop codons or termination codons). 61
codons code for 20 amino acids.
 (1)Specificity or Unambiguity: A specific codon always code
for the same amino acid, and one codon codes for one
amino acid. E.g, UUU=Phe and not for any other aa.
 (2)Universality: Genetic code is the same in all living
organisms. The exception is found in mitochondrial
codons where AUA codes for met, and UGA= Trp instead
of Ile and termination respectively. Also AGA and AGG are
for termination in mitochondria but code for Arg in
cytoplasm
 (3) Redundancy: This is also termed degeneracy. Although
each codon correspond to single amino acid but a single
amino acid can have multiple codons. Only Trp and Met
have one codon each
 (4)Non-overlapping and Non-punctuated: All codons are
independent sets of 3 bases. No overlapping. Codon is
read from a fixed starting point as a continous sequence
of bases, taken 3 at a time. The starting point is extremely
important and this is called Reading Frame
 (5) Non-sense codons: Three codons out of 64 do not
code for any amino acid. They are also called
termination codon or stop codon. UAA, UAG, UGA.
The ribosome stops when any of these codons are
read.
 (6) Initiator codon: AUG is the initiator codon in
majority of proteins. In few cases, it may be GUG.
AUG codes for Met.
 (7) Wobbling phenomenon: The reduced specificity or
stringency (i.e relaxed base-pairing rule) between the
3rd base of the codon and the complementary
nucleotide in anticodon is responsible for wobbling.
 E.g. GGU, GGC and GGA codons can be matched by
anticodon CCI for glycine.
Also CCU, CCC, CCA can be read by anticodon GGI for
proline.
 (8) Codon reading is in triplet of nucleotides in 5’ to
3’ direction.
 Mutation can be well explained using the genetic
code. Mutation can be beneficial, neutral and
harmful.

 (A) Point Mutation:Involves a single nucleotide. This

can be by Transition or Transversion
-Silent mutation (same or similar aa)
-Misense mutation (a different aa)
-Nonsense mutation (stop)

(B) Frame Shift Mutation: Can be by insertion or

deletion

Note: Protein sythesizing machinery of mitochondria is

distinct from that in the cytoplasm. E.g. there only 22
tRNAs in mitochondria, 31 tRNA in cytoplasm
 Translation takes place in the cytoplasm. The mRNA is
translated 5’ to 3’ end. The polypeptide chain grows from
amino terminal to carboxyl terminal end.
 Translation process can be divided into 5 phases:
a) Activation of amino acid b) Initiation
c) Elongation d) Termination
e) Post-translational processing

 (a) Activation of amino acid

Amino acid + tRNA + ATP → Aminoacyl-tRNA + AMP + 2Pi
The enzyme is aminoacyl tRNA synthetase

 The D arm of tRNA recognises the enzyme, the CCA-3’

end of the acceptor arm carries the amino acid. The amino
acid is first activated by ATP, then the COOH group of the
amino acid is esterified with the 3’ OH group of tRNA
Formation of
aminoacyl tRNAs
by aminoacyl tRNA
synthetase.
 (b) Initiation of Synthesis

 The start codon AUG is identified after the marker

sequence(for binding site). In bacteria it is refered to as
Shine-Dalgarno sequence, in mammals it is Kozak
sequence. In eukaryotes, AUG=Met. In Prokaryotes,
AUG= N-formyl-Met.

 GTP, IF-2, Met-tRNA and 40S ribosomal subunit are

complexed to form pre-initiation complex.

 The pre-initiation complex binds with mRNA, facilitated

by the 5’- methylated cap, requiring IF-4A, 4B, 4E, 4F,
4G and hydrolysis of ATP for energy. This is the
initiation complex.

 The 60S ribosomal unit now binds to form the full

assembly of 80S ribosomes. This requires IF-2 and IF-5
and hydrolysis of GTP. Then the Initiation Factors are
released.
 Initiation of
protein
biosynthesis
The complete
ribosome
contains 2
receptor sites
P-site: peptidyl
site receives the
peptidyl tRNA
A-site: aminoacyl
site receives the
new incoming
tRNA carrying the
aa to be added.
The met-tRNA
now at P-site
 (c) Elongation of synthesis

 A new aminoacyl tRNA comes to the A-site, determined by the

next codon in mRNA. EF-1 and GTP are required. EF-1 is
released when tRNA binds to the A-site.

 A peptide bond is formed between the α-NH2 group of the

incoming amino acid in the A-site and the –COOH group of the
peptidyl tRNA in the P-site. This is catalysed by peptidyl
transferase (a ribozyme). The growing peptide chain is now in A-
site.

 The tRNA fixed at the P-site(now carrying no amino acid)is

released from the ribosome(through the E-site) . The whole
ribosome moves over the mRNA a distance of one codon. The
peptidyl tRNA is translocated to the P-site. A-site is ready to
receive another tRNA.
Translocation requires EF-2 and hydrolysis of GTP to GDP.

 These elongation reactions are repeated till the polypeptide

chain synthesis is complete.
Amino end of
polypeptide

E
mRNA 3
P A
site site
5
Amino end of
polypeptide

E
mRNA 3
P A
site site
5 GTP

GDP  P i

P A
Amino end of
polypeptide

E
mRNA 3
P A
site site
5
GDP  P i

P A

P A
 E
mRNA 3
P A
site site
5
GDP  P i

E E

P A P A

GDP  P i

P A
 (d) Termination of Synthesis

 Amino acids are added until the ribosome reaches the

termination codon(UAA, UAG or UGA) on the mRNA.
The A-site remains free because no tRNA has the
corresponding anticodon

 The releasing Factor RF enters the A-site (GTP

dependent) . It hydrolyses the peptidyl chain from the
tRNA at the P-site, releasing the peptide chain. The
80S ribosome dissociates into its component units
(40S and 60S subunits)

 Note: One eukaryotic ribosome can synthesise 5-6

peptide bonds per second. Many ribosomes can work
on the same mRNA simultaneously. Such aggregates
of ribosomes are called polyribosomes or polysomes.
Termination of translation
 1. Proteolytic cleavage: e.g conversion of pro-insulin to insulin

 2. Modification of amino acids: e.g.

i) gamma carboxylation of glutamic acid residues in
prothrombin under the influence of vitamin K
ii) hydroxylation of proline and lysine residues in collagen
with the help of vitamin C
iii) phosphorylation of OH groups of Ser, Thr or Tyr by
kinases in covalent modification of enzymes e.g. glycogen
phosphorylase
iv) cotranslational glycosylation of proteins where
carbohydrates are attached to Ser, Thr, Aspn or Gln as
target signals.

3. Subunit aggregation: e.g. immunoglobulin, hemoglobin and

maturation of collagen

Note: Failure of PTM affect the normal function of many proteins.

E.g. poor cross linking of collagen in scurvy caused by vitamin C
deficiency
 4. Protein folding: ‘Chaperones’ help to produce the
correct spartial arrangement of proteins, by attaching
to nascent polypeptide chains and prevent wrong
folding. Examples of chaperones are heat shock
proteins(HSPs), also called stress proteins. Abnormal
folding of proteins may lead to prion diseases.
 E.g.
in humans: Kuru, fatal familiar insomnia(FFI).
In sheep: scrapie disease.
In cow: bovine spongiform encephalopathy (BSE)
(mad cow disease)

 Chaperonopathies are disorders resulting from ‘sick

chaperones’.
 Protein targetting is the attachement of molecular signals or
‘address’ to proteins after completion of synthesis to ensure
their delivery to their correct destined compartment. It is also
called ‘protein sorting’ or ‘protein localisation’.

 Proteins made for external secretion are synthesised on

rough ER, then delivered to the destined compartment. As the
nascent protein is traversing the inner membrane of ER,
carbohydrate moieties are added at particular regions by
specific enzymes, to act as target signals. This is called
cotranslational glycosylation. Proteins for internal parts of the
cell synthesised on free ribosomes lack this signal sequence.

 The signal peptide is on the amino terminal region of protein.

Proteins also carry ‘address’ that is specific for its correct
destination inside the cell. This is present in the carboxyl
terminal end of proteins. E.g. proteins for peroxisomes
contain peroxisome target sequence (PTS) of 26-36 amino
acids. Proteins for the nucleus contain certain nuclear import
signal sequences.
 (a) Zellweger syndrome: It is due to defective oxidation of
very long chain fatty acids (VLCFA) in peroxisomes.
Peroxisomal enzymes are produced but their entry into
peroxisome is denied. Accumulation of VLCFA in CNS
causes neurological impairment and death in childhood.

 (b) Primary hyperoxaluria: The enzyme alanine glyoxylate

aminotransferase is seen in mitochondria instead of its
normal peroxisomal location. This causes kidney stones at
early age.

 (c) Some familiar cholesterolemia: Is due to deficient

transport signals.

 (d) Inclusion cell disease: Is due to non-entry of normal

enzymes into lysosomes.
 Many antibiotics generally act on the bacteria and are non-
toxic to human beings. This is because mammalian
ribosomes (80S) are different from those of bacteria (70S)

 A. Reversible inhibitors (bacteriostatic)

1. Tetracycline: Binds to 30S subunit of bacteria ribosome
and inhibits attachment of aminoacyl tRNA to the A-site of
ribosome.
2. Chloramphenicol: Inhibits peptidyl transferase activity
of bacterial ribosomes.
3. Erythromycin and Clindamycin: Prevent the translocation
process

 B. Irreversible Inhibitors (bactericidal)

1. streptomycin and all other aminoglycoside antibiotics
bind to 30S subunit of bacterial ribosomes. They cause
misreading of mRNA and at high concentrations,
completely inhibit the initiation complex formation and
totally inhibit protein synthesis.
 Note: These are not suitable for clinical use but useful as
research tools.

1. Puromycin: structurally similar to tyr-tRNA and get attached to

the A-site of ribosome. It acts both in bacteria and mammals.

2. Cycloheximide: inhibits peptidyl transferase in 60S subunit.

Acts only on eukaryotic cells.

3. Diphtheria toxin: this is liberated by corynebacterium

diphtheria, and it causes inactivation of EF-2 by attachment of
ADP to EF-2 and inhibit protein synthesis in mammals.

4. Inhibitors of Transcription will also in turn inhibit translation

process.
Some antibiotics
that act by
inhibiting
protein biosynthesis