The document provides information about protein synthesis and processing. It begins with an overview of the topics to be covered, including ribosome formation, initiation and elongation factors, termination, the genetic code, tRNA aminoacylation, aminoacyl-tRNA synthetases, translational proofreading, inhibitors, and post-translational modifications. It then discusses the machinery of protein synthesis, including transcription, the genetic code, RNA, tRNA identity, aminoacyl-tRNA synthetases, aminoacylation of tRNA, and the ribosome. The mechanisms of initiation, elongation, and termination are explained in detail.

1. Protein synthesis and processing
(Translation: RNA → Protein)
PRESENTED BY:
PRASHANT VC
DEPT OF ZOOLOGY
GUK

2. Unit-IV: Protein synthesis and processing:
Ribosome, formation of initiation complex,
initiation factors and their regulation,
elongation and elongation factors, termination,
genetic code, aminoacylation of tRNA, tRNA-
identity, aminoacyl tRNA synthetase, and
translational proof-reading, translational
inhibitors, Post Translational modification of
proteins. Protein targeting.

3. Syllabus-Reorganized
►Introduction: Protein synthesis and processing
► Machinery of Protein synthesis
1. Transcription - Separate Topic
2. Genetic code - Separate Topic
3. RNA - Separate Topic
4. tRNA-identity
5. Aminoacyl - tRNA synthetase
6. Aminoacylation of tRNA
7. Ribosome
► Mechanism of Protein synthesis
● Initiation: formation of initiation complex, initiation factors and their regulation
● Elongation: elongation and elongation factors
● Termination
► Translational proof-reading
► Translational inhibitors
► Post Translational modification of proteins
► Protein targeting

4.  Genetic information is contained within the nucleus of a cell
 DNA in the nucleus directs protein synthesis but protein
synthesis occurs in ribosomes located in the cytoplasm
 How does a ribosome synthesize the protein required if it does
not have access to DNA?
 The answer lies in an intermediate substance known as mRNA.
 Information is copied from DNA into mRNA, this is
transcription
 mRNA leaves the nucleus and enters the cytoplasm of the cell
 Ribosomes use the mRNA as a blueprint to synthesize proteins
composed of amino acid, this is translation.

6. 1. Transcription - Separate Topic
2. Genetic code - Separate Topic
3. RNA - Separate Topic
4. tRNA-identity
5. Aminoacyl - tRNA synthetase
6. Aminoacylation of tRNA
7. Ribosome

7.  There are three types of RNA:
 mRNA is the “blueprint” for construction of a
protein
 rRNA is the “construction site” where the proteins
are made
 tRNA is the “truck” delivering the proper amino
acid to the site of protein synthesis

9.  The ribosome alone cannot
synthesize the polypeptide
chain
 The correct amino acids must
be delivered to the polypeptide
building site by tRNA
 tRNA look like three-lobed
“cloverleaf” due to base
pairing between
complementary nucleotides on
different regions of each tRNA
molecule causing it to fold

10.  At the end of one lobe of tRNA,
a sequence of three bases called
the anticodon recognizes and is
complementary to the codon of
the mRNA.
 The anticodon sequence is
written in the 3’ to 5’ direction.
 At the 3’ end of the strand is an
attachment site for the
corresponding aa specified by the
mRNA codon.

11.  Aa-tRNA (tRNA molecule
bound to its particular amino
acid) has 2 binding sites;
one is for a specific amino
acid, the other is specific to a
particular anticodon
 When both are in the
enzyme’s active site the
enzyme catalyzes a reaction
that binds the two.

12. ► The correct amino acid is added to its tRNA by a specific
enzyme called an aminoacyl-tRNA synthetase. The process
is called aminoacylation, or charging or activation of aa.
► Since there are 20 amino acids, there are 20 aminoacyl-tRNA
synthetases.
► All tRNAs with the same amino acid are charged by the same
enzyme, even though the tRNA sequences, including
anticodons, differ.
ACTIVATION OF AMINO ACID –
• By enzyme aminoacyl-tRNA synthetase
• Highly specific for each amino acid & its corresponding tRNA
• 2 steps reaction-
► ATP + AMINO ACID  AMINO ACID-AMP + PPi
► AMINO ACID-AMP + t RNA  AMINOACID-tRNA + AMP

13.  Ribosomes are the site of protein
synthesis. A ribosome is a complex that
contains a cluster of different kinds of
proteins and rRNA which are linear
strands of RNA
 The ribosome has binding sites for the
mRNA transcript and the aa-tRNA
molecules.
 Each active ribosome has 3 different
binding sites for tRNA molecules:
P (peptide) site: which holds one aa-tRNA
and the growing chain of amino acids
A (acceptor) site: which holds the tRNA
bringing the next amino acid to be
added to the chain
E (exit) site: which releases the tRNA
molecules back into the cytoplasm

14.  The anticodon of an aa-tRNA
molecule binds to the mRNA
codon exposed in the A site.
 Enzymes catalyze the
formation of a bond between
the last aa on the lengthening
polypeptide and the new aa.
The polypeptide chain is
transferred from the tRNA in
the P site to the tRNA in the A
site.
 The ribosome moves down the
mRNA strand, shifting the
binding site a distance of 3
nucleotides (1 codon), this is
called translocation. A new A
site is exposed as the tRNA that
was in the P site is moved to
the E site and released.

15. ● Initiation: formation of initiation complex,
initiation factors and their regulation
● Elongation: elongation and elongation factors
● Termination

16.  After transcription mRNA exits the nucleus via
nuclear pores and ribosomes bind to mRNA
 Ribosomes synthesize different proteins by reading
the coding sequence on mRNA
 The mRNA is read in triplets of nucleotides each of
which encodes an amino acid

19.  All protein synthesis involves three phases:
initiation, elongation, termination
 Initiation: involves binding of mRNA and initiator
aminoacyl-tRNA to small subunit of ribosome,
followed by binding of large subunit of ribosome
 Elongation: synthesis of all peptide bonds - with
tRNAs bound to acceptor (A) and peptidyl (P) sites
 Termination: occurs when "stop codon" reached

20. ● Requires atleast 11 eukaryotic Initiation Factors (eIFn)
►eIF3 & eIF1A regulates dissociation of ribosomal subunits
► eIF2, GTP & Met-t RNA binds to 40S subunit
 accompanied by eIF1
 ►eIF4F helps initiation complex to bind to 5’ cap of mRNA
● The initiator tRNA is a special one that carries only Met and
functions only in initiation - it is called tRNAi
Met but it is not
formylated (as in prokaryote)

21.  Begins with formation of ternary complex of eIF-2,
GTP and Met-tRNAi
Met
 This binds to 40S ribosomal subunit:eIF-3:eIF4C
complex to form the 40S preinitiation complex
 no mRNA yet, so no codon association with Met-
tRNAi
Met
 mRNA then adds with several other factors, forming
the initiation complex
 ATP is required!
 Proteins of the initiation complex apparently scan to
find the first AUG (start) codon

25. Phosphorylation is the key, as usual
 At least two proteins involved in initiation
(Ribosomal protein S6 and eIF-4F) are
activated by phosphorylation
 But phosphorylation of eIF-2a causes it to bind
all available eIF-2B and sequesters (isolates) it

27. ►Binding of incoming aa-t RNA to
the A-site of ribosome, in the
presence of Elongation Factor
(EF-1) & GTP
► Peptide bond formed between
previous amino acid & newly
entered amino acid by peptidyl
transferase
►Ribosome now moves to next
codon in the mRNA so that
empty P-site get occupied by the
peptidyl-tRNA present in A-site
► Translocation requires EF-2 &
GTP

28.  Translocation of the
ribosome exposes a stop
codon in the A site.
Stop codons do not code
for an aa, there are no
corresponding tRNAs.
 A protein called a
release factor binds to
the exposed A site
causing the polypeptide
to separate from the
remaining tRNA
molecule
 Ribosome falls of the
mRNA and translation
stops

29. ►Termination signaled by one of the
three termination codons
►Only one eukaryotic Release Factor
eRF1 recognize the three stop codons
►eRF3 in combination with GTP,
induces ribosome to release the eRF1
►Polypeptide chain releases & RNAs
leave the ribosome

30. Prokaryotic Translation
1. It occurs on 70 S ribosomes.
2.It is a continuous process as both
transcription and translation occur in cytoplasm
3. mRNA is polycistronic.
4. First amino acid taking part is fmet.
5. Initiation codon is usually AUG,occasionally
GUG or UUG
6. It is a faster process, adds about 20 amino
acids per second.
7. It requires 3 initiation factors IFI. IF2. IF3.
8. After translation, formyl group from first
formylated methionine is removed, retaining
methionine in the polypeptide chain.
9. It requires two release factors RF1 (for UAG
and UAA) and RF2 (for UAA and UGA) in the
termination.
10. mRNA life is short (some seconds to some
minutes) as mRNA is less stable.
Eukaryotic Translation
1. It occurs on 80 S ribosomes.
2. It is a discontinuous process as transcription
occurs in nucleus while translation takes place
in cytoplasm.
3. mRNA is monicistronic
4. First amino acid is met (methionine).
5. Initiation codon is AUG. occasionally GUG
or CUG.
6. It is a slower process that adds one amino
acid per second.
7. It requires a set of more than 9 initiation
factors elF 1, 2, 3, 4A. 4B, 4C, 4D, 5. 6.
8. The whole of initiating methionine is
removed from the polypeptide chain.
9. It requires single release factor eRF 1.
10. mRNA has a life of few hours to few
days. It is quite stable.

31. In eukaryotic mRNAs the 5’ cap structure help define the start
codon. The 40S subunit binds to the cap structure and then
locates the first AUG codon 3’ to the cap structure as the
translation start site.
Shine-Delgarno
sequence
Start signals for the initiation of protein synthesis in (A) prokaryotes and
(B) eukaryotes

32. In procaryotes, there can be multiple ribosome-binding sites (Shine-Delgarno
sequences) in the interior of an mRNA chain, each resulting in the synthesis of a
different protein.
A comparison of the structures of procaryotic and eucaryotic
messenger RNA molecules

33. Formation of the initiation complex.
The complex forms in three steps at
the expense of the hydrolysis of GTP
to GDP and Pi. IF-1, IF-2, and IF-3 are
initiation factors. P designates the
peptidyl site, A, the aminoacyl site,
and E, the exit site. Here the
anticodon of the tRNA is oriented 3’ to
5’, left to right.
Initiation of Prokaryotic Translation

34. Bacterial
Factor Function
IF-1 Prevents premature binding of tRNAs to A site
IF-2 Facilitates binding of fMet-tRNAfMet to 30S
ribosomal subunit
IF-3 Binds to 30S subunit; prevents premature
association of 50S subunit; enhances specificity
of P site for fMet-tRNAfMet

35. Formation of N-Formylmethionyl-tRNAfMet
•A special type of tRNA called tRNAfMet is
used here. It is different from tRNAMet that
is used for carrying Met to internal AUG
codons. The same charging enzyme
(synthetase) is believed to be responsible
for attaching Met to both tRNA molecules.
•Blocking the amino group of Met by a
formyl group makes only the carboxyl
group available for bonding to another
amino acid. Hence, fMet-tRNAfMet is
situated only at the N-terminus of a
polypeptide chain.
•IF2-GTP specifically recognizes fMet-
tRNAfMet, which is brought to only the AUG
start codon at the P site.

36. First step in elongation (bacteria):
binding of the second aminoacyl-tRNA
The second aminoacyl-tRNA enters the
A site of the ribosome bound to EF-Tu
(shown here as Tu), which also contains
GTP. Binding of the second aminoacyl-
tRNA to the A site is accompanied by
hydrolysis of the GTP to GDP and Pi
and release of the EF-Tu•GDP complex
from the ribosome. The bound GDP is
released when the EF-Tu•GDP complex
binds to EF-Ts, and EF-Ts is
subsequently released when another
molecule of GTP binds to EF-Tu. This
recycles EF-Tu and makes it available
to repeat the cycle.

37. Second step in elongation: formation
of the first peptide bond
The peptidyl transferase catalyzing
this reaction is probably the 23S
rRNA ribozyme. The N-
formylmthionyl group is transferred
to the amino group of the second
aminoacyl-tRNA in the A site,
forming a dipeptidyl-tRNA. At this
stage, both tRNAs bound to the
ribosome shift position in the 50S
subunit to take up a hybrid binding
state. The uncharged tRNA shifts
so that its 3’ and 5’ ends are in the
E site. Similarly, the 3’ and 5’ ends
of the peptidyl tRNA shift to the P
site. The anticodons remain in the A
and P sites.

38. Third step in elongation: translocation
The ribosome moves one codon
toward the 3’ end of mRNA, using
energy provided by hydrolysis of
GTP bound to EF-G (translocase).
The dipeptidyl-tRNA is now entirely
in the P site, leaving the A site
open for the incoming (third)
aminoacyl-tRNA. The uncharged
tRNA dissociates from the E site,
and the elongation cycle begins
again.

39. Factor Mass (kD) Function
Elongation Factors
EF-Tu 43 Binds aminoacyl-tRNA and GTP
EF-Ts 74 Displaces GDP from EF-Tu
EF-G 77 Promotes translocation by binding GTP
to the ribosome
Release Factors
RF-1 36 Recognizes UAA and UAG Stop codons
RF-2 38 Recognizes UAA and UGA Stop codons
RF-3 46 Binds GTP and stimulates RF-1 and
RF-2 binding

40. Termination of protein synthesis in
bacteria
Termination occurs in response to a
termination codon in the A site. First, a
release factor (RF1 or RF2 depending on
which termination codon is present) binds
to the A site. This leads to hydrolysis of
the ester linkage between the nascent
polypeptide and the tRNA in the P site
and release of the completed polypetide.
Finally, the mRNA, deacylated tRNA, and
release factor leave the ribosome, and
the ribosome dissociates into its 30S and
50S subunits.

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