MBBS Biochemistry

4. TRANSLATION
 THE LANGUAGEOF NUCLEIC ACID IS
TRANSLATED INTO LANGUAGEOF
PROTEINS
 NUCLEIC ACID HAVE 4 LETTER LANGUAGE
 Protein Synthesis from mRNA

5.  PROTEIN IS IMPORTANT CONSTITUENTOF
EVERY CELL IN BODY
 HAIR AND NAILS MOSTLY MADE UP OF
PROTEINS
 IMPORTANT BUILDING BLOCK OF BONES
,MUSCLES ,CARTILLAGE
 IMPORTANT MACRONUTRIENT
 END PRODUCT OF MOST INFORMATION
PATHWAYS

6. GENETIC CODE
 GENETIC CODE IS SET OF RULES BY WHICH
GENETIC INFORMATION ENCODED IN
GENETIC MATERIAL ( DNA Or RNA ) IS
TRANSLATED INTO PROTEIN
 GENETIC CODE IS INTHE FORM OF CODONS
 CODONS ARE GROUP OF 3 ADJACENT BASES
THAT SPECIFIES AMINO ACID OF PROTEIN
 ACT AS LINK BETWEEN SEQUENCES OF DNA
BASES AND SEQUENCE OF AMINO ACIDS

7. ALMOST UNIVERSAL
 EACH CODON CODES FOR SAMEAMINO
ACID IN ALMOSTALL ORGANISM
 AUG CODES FOR METHIONINE IN ALMOST
ALL ORGANISM
 EXCEPTIONS :-
 UGA CODES FORTYPTOPHAN INSTEAD
SERVING AS STOP CODON
 AUA CODES FOR METHIONINE INSTEAD OF
ISOLEUCINE

9. WOBBLE HYPOTHESIS
 BY FRANCIS CRICK
 THIS EXPLAINWHY MULTIPLE CODON
CODE FOR SINGLE AMINO ACID
 THIS EXPLAINS DEGENERACYOF GENETIC
CODE
 DIFFERENT CODON CODE FOR SAME
AMINO ACID BUT DIFFRENCE LIE AT 3rd base
OF mRNA
 Alanine coded by :- GCU , GCC , GCA , GCG

10. 1st BASE OF ANTICODON DETERMINES THE NO OF
CODON WHICH CAN BE READ BY tRNA AS 1st
BASE OF ANTICODON WOBBLE IN FOLLOWING
MANNER
1st BASE OF ANTICODON
U
G
I
A
C
 3rd BASE OF CODON
 A , G
 U , C
 U , C , A
 U
 G

11.  THE CODON THAT DIFFER IN EITHER OF FIRST
2 BASES REQUIRE DIFFERENT tRNA
 eg:- CUU & CUA --- LEUCINE
 CCU &CCA ---PROLINE
IMPORTANCE OF HYPOTHESIS
ATTHE WOBBLE BASE CODON PAIR ONLY
LOOSELY WITH ITS CORRESPONDING BASE IN
ANTICODON , IT PERMITS RAPID DISSOSCIATION
OF tRNA FROM CODON DURING PROTEIN
BIOSYNTHESIS IF ALLTHREE BASEA OF CODON
ENGAGED IN STRONG WATSON CRICK PAIRING
WITHTHREE BASES OF ANTICODON SO tRNA
DISSOCIATETOO SLOWLY ANDTHIS WOULD
LIMIT PROTEIN SYNTHESIS

12. TRANSLATION
REQUIREMENT
OF
COMPONENTS
POSTTRANSLATION
MODIFICATIONS
PROTEIN
SYNTHESIS
PROPER
ACTIVATION
OF AMINO
ACIDS

13. 1. Amino acids
2. Ribosomes
3. mRNA
4. tRNA
5. Energy sources
6. Protein factors

14. - Proteins are polymers of amino acids
- 20 amino acids found in protein structure
-Absence of any essential amino acid in diet will make
its mRNA codon useless

15. 2. RIBOSOMES
- Huge complex structure of protein and rRNA
- Each ribosome composed of 2 subunits- larger & smaller
- Three sites – A site, P site and E site
- A site considered as acceptor site and binds with
aminoacyl tRNA
- P site considered as donor site and binds with peptidyl
tRNA
- E site for Exit of empty t-RNA

17. Types of Ribosomes
Eukaryotic
 Larger
 Whole ribosome is
80s
 60s + 40s = 80s
Prokaryotic
 Smaller than
eukaryotic
ribosomes
 Whole ribosome is
70s
 50s + 30s =70s

19. POLYRIBOSOME
I. Group of ribosome bound to mRNA molecule
- Many ribosomes can translate the same mRNA molecule
simultaneously
- Increases the efficiency of utilization of mRNA

21. 3. MRNA
-it is a template for protein synthesis
-Translated from 5’ to 3’end
-Polypeptide chain is synthesized from amino
terminus to carboxyl terminus

22. Types of mRNA
PROKARYOTIC mRNA
 Many sites for
initiation
&termination
 Polycistronic
 Translation of
MRNA begins while
it still is being
transcribed
 Life span is very
short
EUKARYOTIC mRNA
 Only one site for
initiation &
termination
 Monocistronic
 Translation and
transcription doesn’t
take place
simultaneously
 Life span is longer,
hence metabolically
stable

25. 4. TRNA
-Smallest RNA
-Adapter RNA
-Structure
CLOVER LEAF MODEL (2D)
L shaped (3D)

27. 5. ENERGY CONSUMPTION
Formation of one peptide bond include the hydrolysis of
4 high energy phosphate bond
Charging of tRNA requires hydrolysis of an ATP to AMP
Entry of aminoacyl tRNA into A site – requires hydrolysis of a
GTP to GDP
 translocation of peptidyl tRNA – requires hydrolysis
of GTP to GDP and phosphate

28. When the tRNA is carrying an amino acid, it is
referred to as being charged and the amino acid
as being activated.
The carboxy group of an amino acid is
esterified to the hydroxy group of ribose unit at
the 3’ end of tRNA chain.
20 different enzymes known as
aminoacyl tRNA synthases.

30.  Initiation of a polypeptide chain a
prokaryotes requires :
1. ribosome
2. mRNA
3. initiation Met-tRNA met
4. initiating factor (eIF)

31. Ribosome dissociation
 In this step ribosome dissociated into 30s and 50s
subunit
 30s subunit combines with IF3 IF2 and IF1
 This delays as re association soon with 50s subunit
2 FORMATION OF 30S INITIATION COMPLEX
30S ribosome consist of unique sites known as E P A
 Now IF 3 2 1 attached to there respective sites
 Now mRNA binds with 30s subunits containing
start and stop codon

32. Initiation in a polypeptide chain in
eukaryotes requires:
 Dissociation of the ribosome into its 40s
and 60s subunits
 Binding of a ternary complex consisting of
the initiatior methionly-tRNA , (met-
tRNA),GTP,and eIF-2 to the 40s ribosome
to form the 43s preinitiation complex
 Binding of mRNA to the 40s preinitiation
complex to form the 48s initiation complex
 Combination of the 48s initiation complex
with the 60s ribosomal subunit to form the
80s initiation complex.

33.  There is a cross stocky between the ribosome &
mRNA in 30s subunit containing a particular purine
rich DNA sequence called “shine Dalgarno sequence
“(s-d-s GGAGGAAU) in 16-rRNA (Prokaryotes)
 mRNA which is complementary to the S-D-S now
pass with it which is upstream to starting codon
 In Eukaryotes No s-d-s, identify AUG and binding of
ribosome

35. 2. Formation of the
43s preinitiation complex
 The first step in this process involves the binding of
GTP by eIF-2
 This binary complex then binds to met tRNA ; a
tRNA specifically involved in binding to the initiation
codon AUG (there are two tRNAs for methionine ,
one specifies methionine for the initiator codon, the
other for internal methionines .each has a unique
nucleotide sequence;both are aminoacylated by the
same methionly-tRNA synthetase.)
 This ternary complex binds to the 40s ribosomal
subunits to form the 43s preintiation complex,which
is stabilized by association with eIF-3 nd eIF-1A.

36. Formation of 48s initiation complex
 The binding of mRNA to the 43s preinitiation
complex forms 48s initiation complex
 The 5’ terminals of most mRNA molecules in
eukaryotic cells are capped by methylguanosly
triphosphate, which facilitates the binding of mRNA
to the 43s pre-initiation complex
 The association of mRNA with 43s initiation complex
requires cap binding protein (CBP),eIF-4F,andATP.
 eIF-4F complex is formed by the association of eIF-
4G ,eIF-4A with eIF-4E.
 The association of mRNA with the 43s initiation
complex occurs by hydrolysis of ATP.

37. Recognition of initiation
codon
 The ribosomal initiation complex scans the
mRNA for the identification of appropriate
initiation codon . 5’ – AUG is the initiation
codon and its recognition is facilitated by a
specific sequence of nucleotides surrounding
it.
 This marker sequence for the identification of
AUG is called as KOZAK CONSENSUS
SEQUENCES

38. Formation of 80s initiation
complex
 Combination of the 48s initiation complex with
60s ribosomal subunits forms 80s initiation
complex.
 The binding involves the hydrolysis of GTP
(bound to eIF-2 ).This step is facilitated by the
involvement of eIF-5.
 As the 80s complex is formed , the initiation
factors bound to 48s initiation complex are
released.
 These factors are then recycled at this stage ,the
Met-tRNA is on the P site of the ribosome and is
now ready for the elongation process.

39. Regulation of initiation
 The eIF-4F, a complex formed by the
assembly of three initiation factor controls
initiation , and thus the translation process.
 eIF-4E , a components of eIF-4F is primarily
responsible for the recognition of mRNA
cap and this step is the rate- limiting in
translation.

42. Elongation
Stepwise addition of amino acids to the growing
protein chain.
The order of amino acids is specified by the sequence
of codons in the mRNA.
Protein factors, eEFs (eukaryotic elongation factors),
are necessary to speed up the elongation cycle .

43. OR
 elongation a cyclic process on the
ribosome in which one amino acid at a
time is added to the nascent peptide chain
.The amino acid sequence is determined
by the order of the codons in the mRNA
elongation involves following steps :

45. Steps
1 ) Amino acid-containing tRNA
molecules (aminoacyl-tRNAs, aa-
tRNA) are picked up by elongation
factor eEF- in the presence of GTP.

46.  2).The formed complex enters
the empty A-site on a ribosome
carrying an initiator Met-tRNAi
or a peptidyl-tRNA.

47.  3). On the ribosome, the
anticodon of the incoming
aminoacyl-tRNA is matched
against the mRNA codon
positioned in the A-site

48. 4)When the right aminoacyl-tRNA enters
the A-site the growing polypeptide in
the P-site is almost immediately linked to
the new amino acid in theA-site via a
peptide bond.
 Formation of the peptide bond is catalysed
by the ribosome itself.
 The reaction leaves an empty tRNA in the
ribosomal P-site and the new peptidyl-
tRNA in the A-site.

49.  5). Ribosome moves one codon
forward on the mRNA.
 Simultaneously, the empty tRNA is
displaced from the P-site to the E-
site as the peptidyl tRNA is
translocated from the A-site to the
P-site.
 The process is facilitated by
elongation factor eEF-2 and GTP

50.  6). After the translocation, the
peptidyl-tRNA is positioned in the
P-site and the next codon on the
mRNA is made available for
interaction with a new aminaoacyl-
tRNA in the A-site.

53. INHIBITORS
Translation can be inhibited by antibiotics.
Majority of antibiotics interfere with the
bacterial protein synthesis and are harmless
to higher organism.
Such antibiotics can be used as therapeutic
drugs.

54. They generally act only on bacteria and are nontoxic to human
beings. This is because mammalian cells have 80S ribosomes,
while bacteria have 70S ribosomes.
Reversible Inhibitors in Bacteria
These antibiotics are bacteriostatic.
Tetracyclins inhibit attachment of aminoacyl tRNA to the A site of
ribosomes.
Chloramphenicol inhibits the peptidyl transferase activity of bacteria.
Erythromycin and clindamycin prevent the translocation process.
Irreversible Inhibitors in Bacteria
These antibiotics are bactericidal.
Streptomycin binds to 30S subunit of bacterial ribosomes. They cause
misreading of mRNA and totally inhibit protein synthesis.
Inhibitors of Protein Synthesis

55. Inhibitors of Protein Synthesis in Eukaryotes
Puromycin is structurally similar to tyrosine-tRNA and gets
attached to the "A" site of the ribosome. So, the incomplete peptide
is released.
Cycloheximide inhibits peptidyl transferase in 60S subunit.
Diphtheria toxin, liberated by the bacteria, Corynebacterium
diphtheria, causes inactivation of EF-2 by attachment of ADP to EF-2
and consequent inhibition of protein biosynthesis in mammalian
systems.
Inhibitors of Protein Synthesis

56. Differences between Prokaryotic and Eukaryotic Translation
Feature Prokaryotes Eukaryotes
Ribosomes 30S small subunit and
50S large subunit
associate to form 70S
ribosome
40S small subunit and 60S
large subunit together form
the 80S ribosome
Initiation Translation starts before
transcription is
completed. No
mRNA processing
Transcription and
translation are well
separated and the
processed mRNA is
translated
Consensus
sequences
Initiating codon AUG is
recognised by the Shine
Dalgarno sequence
Initiating codon AUG is
recognised by Kozak
sequence
Initiator
tRNA
The initiating tRNA
carries formylated
methionine
The initiating tRNA carries
methionine

57. Differences between Prokaryotic and Eukaryotic Translation
Feature Prokaryotes Eukaryotes
Formation
of initiation
complex
Initiation factors and
GTP hydrolysis are
required for the binding
of the small ribosomal
subunit to the mRNA
Several IF s and GTP
hydrolysis are required for
the formation of the
initiation complex.
Peptide
bond
formation
The peptidyl transferase
ribozyme is in the 23S r
RNA
Peptidyl transferase activity
in the 28S rRNA of the large
subunit.
Post-
translationa
l events
Mainly co-translational
modifications
Mainly post-translational
modification, sorting, export
and localisation
Inhibition Antibiotics can inhibit
different stages of
translation
Toxins like diphtheria, ricin,
puromycin and
cycloheximide

58. OTHERS:
LEIGH’S SYNDROME – Movement
disorders
MYOCLONIC EPILEPSY- Dementia etc.
TERMINATION OF TRANSLATION

59. POST-
TRANSLATIONAL
MODIFICATIONS

60. DNA RNA
protein
The protein synthesised in translation are ,as
such not functional.
Many changes are takes place in polypeptides
after the protein synthesis is completed to
convert them into their active form.

64.  Protein folding is a physical process by
which a polypeptide folds into it’s
characteristic & functional 3 dimensional
structure from random coil.

65.  Each protein exists as an unfolded polypeptide or
random coil when translated from a sequence of
management to a linear chain of amino acids.
 Folding of a newly synthesized polypeptide in the
cellular environment required the assistance of so
called molecular chaperone protein.

66. Chaperones- chaperones are heat shock
proteins.
 They facilitate & favour the interaction on
polypeptide surface to finally give the specific
conformation of a protein.
 Chaperones can reversibly bond to hydrophobic
region of unfolded proteins and folding
intermediates.

67. Chaperones categorised into tow major
groups
 Hsp70 System –These proteins can
bind individually to the substrate
(protein) & help in the correct
formation of protein folding.
 Chaperonin System –they form a
structure into which the folded
protein are inserted.

68. Trimmingbyproteolyticdegradation
Many proteins are synthesised as the
precursor which are much bigger in size
than active protein.
Protein(bigger)-unwanted portion= active
protein
 Some portion of precursor molecules
are removed by proteolysis to
liberate active protein.
 This process is referred to as
trimming.
 Example –formation of insulin from proinsulin
Formation of trypsin from trypsinogen

69. Intein Splicing
 Inteins- inteins are intervening
sequence in certain proteins.
 These are comparable to introns in
mrnas.
 Inteins have to be removed and
exteins ligated in appropriate
order for the protein to become
active.

71. Covalent modifications
 Phosphorylation –the hydroxyl
group containing amino acid of
proteins namely serine, threonine
&tyrosine are subjected to
phosphorylation.
 The phosphorylation may either
increase or decrease the activity
of protein.

72.  Hydroxylation- amino acid proline
and lysine are converted to
hydroxyproline and hydroxylysine
respectively during collagen
formation.
 Hydroxylation occurs in er.
 Glycosylation –the complex
carbohydrates moiety is attached to
amino acid & threonine or to
aspergine, leading to the synthesis
of glycoproteins.

73. What if ptm doesn’t take place???
 The inactive precursor molecules will not be
able to perform their targeted functions.
 Stability of protein will be lost.
 They will not be able to perform biochemical
activity and so on.

74. Protein misfolding
 “the failure of a protein to fold properly,
generally loads to it’s rapid degradation.
 Protein misfolding often results in the
formation of prions (pretentious infectitious
agents) which have been implicated in many
diseases.
 Example – 1)mad cow disease
 2)alzheimer's disease

75. PROTEIN TARGETING
 Proteins carry an ‘address’ that is specific
for its correct destination in the cell.
 Present at Carboxy terminal of proteins.
 Diseases due to defective protein
targeting:
1.) Zellweger Syndrome (peroxisomes)
2.) Hyperoxaluria (peroxisomes)
3.) Inclusion cell disease (lysosomes)

76. Zellweger syndrome is due to defective oxidation of very long
chain fatty acids (VLCFA). Here the correct “address” is not
printed on the protein packet; so that it could not be delivered to the
correct locality. Peroxisomal enzymes are produced; but their entry
into peroxisome is denied. This leads to insufficient oxidation of
VLCFA. Accumulation of VLCFA in CNS causes neurological
impairment and death in childhood.
Another example is primary hyperoxaluria, which causes kidney
stones at an early age. The defect is due to protein targeting defect and
the enzyme alanine glyoxalate aminotransferase is seen in
mitochondria, instead of its normal peroxisomal location.
Familial hypercholesterolemia is due to deficient transport signals.
Inclusion cell disease is due to non-entry of normal enzymes into
lysosomes. Mannose-6-phosphate which is the marker to target
enzymes to lysosomes; is absent in this disease.
Defects in Protein Targeting

77. Some of the mitochondrial protein synthesis is under the
control of mitochondrial DNA. The mtDNA has information
for synthesis of components of electron transport chain. However,
most of mitochondrial proteins are encoded by nuclear DNA and
synthesised in the cytoplasm. Important proteins of the outer
membrane of the mitochondria are synthesised under the influence
of nuclear DNA.
Mitochondria are similar to bacteria than mammalian cells. This
fact supports the theory that mitochondria are derived from
prokaryotes symbiotically adapted to multicellular organisms.
Mitochondrial DNA

78. Feature Eukaryotes
(mammalian
cells)
Prokaryotes
(bacteria)
Mitochondria
DNA Open Circular Circular
Ribosomes 80S 70S 70S
tRNA (No.) 31 22 22
Initiating
amino acid
Methionine Formyl
methionine
Formyl
methionine
Effect of
tetracyclin
Not affected Inhibited Inhibited
Comparison of translation in eukaryotes and prokaryotes.
Mitochondria are similar to prokaryotes.

79. Maternal inheritance: Since the mitochondria are inherited
cytoplasmically, the mtDNA is inherited from the mother.
There are hundreds of copies of mtDNA in each cell (nuclear DNA
has only 2 copies).
If mutation occurs in mtDNA, the daughter cells may inherit the
mutant or normal mtDNA (Heteroplasty)
High mutation rate.
Accumulation of mutations in mtDNA may be responsible for age
related degenerative diseases.
Mitochondrial DNA

80. Syndrome Features
Leber's hereditary
neuropathy (LHON)
Complex I defect; blindness,
cardiac conduction defects
Myoclonic epilepsy ragged
red fiber disease (MERRF)
Myoclonic epilepsy, myopathy,
dementia
Mitochondrial
encephalopathy lactic
acidosis stroke like
episodes (MELAS)
Complex I defect; lactic acidosis,
strokes,myopathy, seizures,
dementia
Leigh's syndrome Complex I defect; NDUFS gene
defect; movement disorders
OXPHOS (Oxidative Phosphorylation) Diseases

81. Micro-RNAs or miRNAs are about 21–25 bases in length. They
have RNA hairpin structure (showing internal hybridization to
make it two strands), and are called short hairpin RNA (shRNA). In
cytoplasm, out of the two strands, one is broken by dicer nuclease. The
selected strand is called the guide strand, which is incorporated into the
RNA-induced silencing complex (RISC) to form functional silencer of
mRNA.
The micro-RNAs bind to matching pieces of messenger RNA, turn it into a
double strand and keep it from doing its job. The process effectively blocks
the production of the corresponding protein, causing translation arrest.
More than 2,500 human miRNAs have been identified.
Circulating miRNA is a rich source for potential disease biomarkers.
Micro -RNA

82. Short double-strand RNA, about 21–25 bases, would silence
the corresponding gene. The RNA interference (RNAi) is a
faster way to turn off genes. Both RNAi and micro-RNA result in
decreased levels of functional proteins in the cells.
Exogenous manipulation of RNAi is being explored as a powerful
method of silencing disease-causing genes in incurable neurological
disorders.
Interfering RNA or RNAi or siRNA

83. Principle of antisense therapy.

84. References
 Textbook of Biochemistry, vasudevan
 Textbook of Biochemistry, lippincott