TRANSLATION
Translation = Protein Synthesis
“An understanding of protein synthesis, the
most complex biosynthetic process, has been
one of the greatest challenges in
Biochemistry” − Lehninger
TRANSLATION is a part of the overall process
of gene expression.
It represents the process of decoding the genetic
information present in the form of Nucleotide
Triplets in the messenger RNA into a sequence of
Amino Acids in the Polypeptide according to the
rules specified in the Genetic Code using mRNA
as the template to guide the protein synthesis.
Translation takes place in the cytosol where
functional ribosomes are located.
The Discovery of Genetic Code
1953- DNA Double helix- James Watson , Francis Crick, Maurice Wilkins
George Gamov----1955 –Proposed triplet code in DNA is directly converted into amino
acid sequence
Crick & Watson- in RNA Tie Club proposed that the triplet information is carried by an
intermediary molecule called Adapter that interacts with amino acids may be involved
in carrying the code (tRNA)
The Discovery of Genetic Code: 1968-Nobel prize in Physiol. Med.
Marshal Nirenberg------ in vitro translation of Poly (U) –UUUUUU- sequence RNA
yielded Poly Phenylalanine.
Severo Ochoa-----Poly (A) coded for Lysine, Poly (C) -coded for Proline
Robert Holley---- Purified tRNA, determined the first nucleotide sequence of an RNA
and predicted the clover leaf structure. This discovery led to the adaptor RNA (tRNA)
carrying the amino acids corresponding to codons.
Hargobind Khorana-----He synthesized mixed sequence RNA oligonucleotides and led
to the cracking the complete genetic code. Dinucleotide repeat UCUCUCUC coded
for Ser and Leu, trinucleotide repeat UAC UACUACUAC (UAC,ACU, CUA)-coded for
Tyr, Thr, Leu and so on.
The Ribosome
Nobel Prize in Chemistry 2009:
Thomas A Steitz, Venkataraman Radhakrishnan & Ada E. Yonath
Components required for translation:
Translation Machinery: The Ribosomes
tRNA
mRNA
Amino acids
Aminoacyl tRNA synthetases I &II
Mg2+
ATP, GTP
Initiation, Elongation & termination factors
Several other proteins
Central Dogma
DNA
TRANSCRIPTION
mRNA
TRANSLATION
PROTEIN
Coding/sense strand CENTRAL DOGMA
5’-tcgactac ATG CTC CGC AAA TTT CCC GGG TGA atcagctacct- 3’ DNA
3’-agctgatg TAC GAG GCG TTT AAA GGG CCC ACT tagtcgatgga-5’
Template/antisense strand Transcription
5’-ucgacuac AUG CUC CGC AAA TTT CCC GGG TGA aucagcuaccu- 3’ mRNA
Decoding/Translation
5’- AUG CUC CGC AAA TTT CCC GGG TGA - 3’
Met Leu Arg Lys Phe Pro Gly Ter
Rate of translation in Prokaryotes ranges from 1000-4000 aas /min
In eukaryotes it is much less , ranges from 200- 400 aas/min
GENETIC CODE
Genetic code consists of sequence sets of 3 nucleotides,
each set corresponding to a specific amino acid.
Characteristics of Genetic Code
specific / unambiguous
Triplet codons
Universal
Nonoverlapping & Commaless
Degenerate & Redundant : More than one codon for
several amino acids
Stop/ Termination/ Nonsense codons
Messenger RNA (mRNA)
mRNA is a transient intermediate between DNA and
Protein in the pathway of information flow from DNA to
protein.
It is derived by copying the deoxyribonucleotide
sequence information present in DNA into Ribonucleic
acid (RNA) sequence by the process called
TRANSCRIPTION.
The nucleotide sequence information present as
contiguous nucleotide triplets called CODONS in mRNA
is decoded into corresponding amino acids by the
ribosome by the process called TRANSLATION.
Reading Frame & Open Reading Frame
Reading Frame in Genetic Code
Reading frame is a contiguous set of codons corresponding to the
amino acid sequence of a polypeptide or no known polypeptide.
Any RNA can have three possible reading frames
5’--- AUC GAC AGC AAA CAG UUU GGG TGG CCA--- 3’
5’-- A UCG ACA GCA AAC AGU UUG GGT GGC CA--- 3’
5’- AU CGA CAG CAA ACA GUU UGG GTG GCC A-- 3’
What is Open Reading Frame (ORF)/Protein coding region ?
The uninterrupted nonoverlapping string of codons starting from the
initiator codon up to and including the termination codon, encoding a
polypeptide constitutes an ORF.
The original ORF can change the frame due to insertion /deletion
mutations or ribosome-mediated frame shift.
Consequences of altered nucleotide sequence on
protein sequence/structure & Function
Possible outcomes of Single nucleotide mutations/Point mutations:
1. Silent mutations ------ no change in amino acid sequence in protein
2. Missense mutations ------ altered amino acid sequence in protein (loss or gain of
function or altered structure)
3. Nonsense mutation ------ Premature termination of protein chain (Truncated
protein & loss of function)
4. Frame-shift mutation ------ Altered protein sequence from the point of one or two
nucleotide deletion/insertion (Altered structure & function)
CCA Missense mutation UCA Silent mutation UCU
(Pro) (Ser) (Ser)
Nonsense
mutation
UAA
(Ter)
TRANSFER RNA (tRNA)
tRNA functions as an adaptor molecule
between the codon and the amino acid for
which the codon specifies.
There is at least one tRNA for each amino
acid and each tRNA recognizes a specific
codon
Each tRNA contains an anticodon that is
complementary to the codon in mRNA.
tRNAs contain unusual / modified bases
such as thymidine,dihydrouridine
pseudouridine, methyl guanine,
hypoxanthine etc.
Modified bases are not essential, but they
play roles in specific recognition of
codons, aaRS recognition, stability etc and
Anticodon CAU
their absence in tRNAs results in reduced
3’-codon-----GUA-----mRNA-5’
growth.
tRNA
7 bps in Acceptor stem
A 3’ 5 bps in TC Arm
C
C 5 bps in Anticodon Arm
5’ 4 or 5 bps in D Arm EXCEPTIONS:
Acceptor In some mitochondrial tRNAs of
G-U, G-, or A- bp
mammals, flagellates and
nematodes, gross deviation in D-
D-loop/Arm TC-loop/arm stem loop or lack of D-stem loop
altogether is observed.
These are functional & might
Extra Arm: The most represent remnants of evolution
variable feature in tRNAs. of tRNA structure- i.e.,
In 75% tRNAs it is 5-6 nt & MINIMAL ADAPTOR
Anti-codon loop/arm
in the rest, it is 15-21 nt.
The questions:
How tRNAs that are very similar in structure are discriminated by different
[Link]?
What is the role of modified bases? (25% in human tRNAs).
Identity determination/anti-determination.
Restriction of codon recognition/expansion.
2
4
tRNAs
Isoacceptors: tRNAs that accept the same amino acid
Cognate tRNAs: tRNAs recognized by an aa-tRNA synthetase (aa-
RS).
Advantages & disadvantages of highly similar structure of tRNAs.
Helps in processing by common enzymes.
Poses a problem for recognition by aa-RS for differentiation
between different tRNAs.
tRNA- aa-RS recognition
Distinguishing features in tRNA are primarily located in the
anticodon loop & acceptor stem and a few bases in T- and D –
stems.
2nd genetic code = tRNA recognition by aa-RS
Aminoacylation of tRNA: Charging of tRNA
→ [Link] + Ppi
2+
Enzyme + aa + ATP Mg
tRNA + [Link]-AMP → [Link]-tRNA + AMP + Enzyme
Errors due to misincorporation of aas into proteins = 1 in 104
Errors due to mis-selection of tRNAs = < 1 in 107
Errors due to mis-selection of aas i.e., similar aas = 5 in 105
A.A. selection limits the precision of aminoacylation.
Specificity of charging of cognate tRNAs by aa-RS is achieved by:
1. Binding specificity of tRNA
2. Rate of catalysis.
Differences in binding affinities for cognate & noncognate tRNAs 102
Differentiation at tRNA binding to aa-RS is weak.
Rate of catalysis is 4 orders of magnitude higher for the cognate species.
Specificity of aa-acylation depends primarily on rate of catalysis.
tRNAs
Advantages & disadvantages of highly similar structure of tRNAs.
Helps in processing by common enzymes.
Poses a problem for recognition by aa-RS for differentiation
between different tRNAs.
tRNA- aa-RS recognition
Distinguishing features in tRNA are primarily located in the anticodon loop &
acceptor stem and a few bases in T- and D – stems.
Anticodon Recognition Acceptor Stem Recognition
tRNAPhe tRNAAla
tRNAArg G3-U70 → Para wobble bp
tRNAMet Introduction of wobble bp
tRNAGlu into the acceptor stem of tRNALys
tRNAVal results in conversion to tRNAAla
tRNAGln tRNAAla identity is associated with a distortion
in the acceptor stem helix due to wobble bp.
Mini helix having G3-U70 + TC arm → functions as tRNAAla
Para codon : G3-U70
tRNA STRUCTURE
tRNA & aa-RS Recognition
The features necessary for tRNA
identification so that they can be
differentiated by aa-RS & can be
aminoacylated correctly are called
Recognition Elements.
If they are altered,the tRNA is
aminoacylated at a slower rate or
mischarged.
Each set of cognate tRNAs contains a
set of identity elements that are
common.
tRNA recognition by aa-RS constitutes the 2nd genetic cade, which states that the
Code is imprinted into the structure of an aa-RS which matches structural feature of
Its cognate tRNAs.
Crick’s Wobble Hypothesis: 4 relationships
Allowed wobble base pairing combinations at
3rd codon-anticodon position
Wobble allows a tRNA to recognize more than one codon. When
several codons specify a single amino acid , the different codons
generally differ at the 3rd base.
Wobble Hypothesis
Inosine
RIBOSOMES
Ribosomes are highly organized macromolecular complexes of RNAs and
Proteins, that decode the nucleotide sequence in the mRNA into amino acid
sequence in the polypeptide.
They are composed of about 40% of proteins and 60% ribosomal RNA (rRNA).
Each ribosome contains 2 subunits- one small subunit and one large subunit.
The large and small subunits undergo association and dissociation during the
start and end of each translation cycle to initiate new round of translation.
The Large subunit contains Peptidyl transferase centre that catalyses peptide
bond formation.
The small subunit binds mRNA and contains the decoding centre in which the
charged tRNAs read or decode codons in mRNA into amino acids in the
polypeptide.
Peptidyl tRNA site
Functions of ribosomal subunits and Ribosomes
The Large subunit contains the peptidyl transferase site formed by the ribosomal
RNA that is responsible for peptide bond formation.
The small subunit binds mRNA and contains the decoding centre in
which the aa-tRNAs decode the codons.
Three tRNA binding sites (A, P and E) are formed at the interface of
the large and small subunits upon their association in presence of
mRNA.
1. A site: Aminoacyl tRNA-binding site
where the first aminoacyl tRNA binds.
2. P site: Peptidyl tRNA binding site.
3. E site: Exit site for the exiting tRNA
after transfer of the polypeptide chain
to the incoming peptidyl tRNA at the P site.
Functional ribosomes & subunits
are localized in cytoplasm.
Functionally competent Ribosomes
Size comparison of ribosomes and tRNAs
Size comparison shows that ribosomes can easily
accommodate the tRNAs.
A 35-nucleotide mRNA fragment is protected from RNase digestion
by the complete ribosome.
Aminoacyl tRNA synthetase (aa-RS) specificity.
One synthetase for one amino acid and the synthetase
can recognise multiple tRNAs corresponding to the same
amino acid. The tRNAs recognised by one aa-RS are called
cognate tRNAs.
Two types of aa-RSs exist Based on the site on the terminal
ribose of tRNA where the amino acid is attached to the
tRNA.
tRNAs
Isoacceptors: tRNAs that accept the same amino acid
Cognate tRNAs: tRNAs recognized by an aa-tRNA synthetase (aa-
RS).
Advantages & disadvantages of highly similar structure of tRNAs.
Helps in processing common enzymes.
Poses a problem for recognition by aa-RS for differentiation
between different tRNAs.
tRNA- aa-RS recognition
Distinguishing features in tRNA are primarily located in the
anticodon loop & acceptor stem and a few bases in T- and D –
stems.
tRNA & aa-RS Recognition
The features necessary for tRNA
identification and differentiation by
aa-RS & can be aminoacylated
correctly are called Recognition
Elements.
If they are altered, the tRNA is
aminoacylated at a slower rate or
mischarged.
Each set of cognate tRNAs contains a
set of identity elements that are
common.
tRNA recognition by aa-RS constitutes the 2nd genetic cade, which states that the
Code is imprinted into the structure of an aa-RS which matches structural feature of
Its cognate tRNAs.
2nd genetic code = tRNA recognition by aa-RS
tRNA- aa-RS recognition
Distinguishing features in tRNA are primarily located in the anticodon loop &
acceptor stem and a few bases in T- and D – stems.
Anticodon Recognition Acceptor Stem Recognition
tRNAPhe tRNAAla
tRNAArg G3-U70 → Para wobble bp
tRNAMet Introduction of wobble bp
tRNAGlu into the acceptor stem of tRNALys
tRNAVal results in conversion to tRNAAla
tRNAGln tRNAAla identity is associated with a distortion
in the acceptor stem helix due to wobble bp.
Mini helix having G3-U70 + TC arm → functions as tRNAAla
Para codon : G3-U70
Charging of tRNA/aminoacylation of tRNA
Enzyme + aa + ATP → [Link] + PPi
tRNA + [Link]-AMP → aa-tRNA + AMP + Enzyme
Initiation of protein synthesis by Formyl-Met-initiator tRNA in Bacteria
DIFFERENT STAGES OF TRANSLATION
1. Charging of tRNAs with cognate amino acids
2. Initiation:
Prokaryotes: Direct Binding of mRNA to small subunit
and loading of fMet-tRNAf by initiation factors at P site at AUG codon and
recruitment of large subunit. SD seq. 5’-UAAGGAGG-3’
16S rRNA 3’seg 3’-AUUCCUCC-5’
Eukaryotes: Binding of Initiation factors-bound mRNA to the small ribosomal
subunit and loading of Met-tRNAi by initiation factors at P site. Scanning of
mRNA from Cap to the AUG codon and recruitment of large subunit.
Pioneer round of translation promoted by nuclear Cap-binding complex (80 kDa
an 20 kDa subunits) and eIF4F
3. Elongation: codon specific elongator aa-tRNAs loaded at A site and the
polypeptide chain from tRNA at P site transferred to the new tRNA at A site and
movement of peptidyl tRNA from A to P site.
4. Termination: Binding of termination factors at A site when the STOP codon is
reached at the A site and release of polypeptide
Initiation ("beginning"): in this stage, the 2 ribosome subunits are
assembled together with the mRNA and the first Met-tRNA, and
translation begins.
•Elongation ("middle"): in this stage, amino acids are brought to the
ribosome by tRNAs and linked together by peptide bonds to form a
polypeptide chain.
Termination ("end"): in the last stage, the finished polypeptide is
released from the ribosome to go and do its job in the cell.
Components required for different stages of protein
synthesis in E. coli
1. Charged of tRNAs:
20 amino acids, 20 amino acyl tRNA synthetases
32 tRNAs; ATP; Mg2+
2. Initiation:
mRNA with SD sequence and AUG codon,
N-formayl Met-tRNA
30S and 50S ribosomal subunits
Initiation factors-IF1, IF2 and IF3; GTP, Mg2+
3. Elongation:
Functional 70S ribosome assembled on the mRNA
All the aminoacyl tRNAs specified by the codons
Elongation factors EF-Tu, EF-Ts and EF-G; GTP, Mg2+
4. Termination:
Termination codon in mRNA
Termination/Release factors RF-1, RF-2 and RF3
5. Folding, Posttranslational modification, processing:
Ribosomes, chaperones, ER, ER-enzymes,
modifying enzymes, ATP,
Eukaryotic mRNAs do not have SD sequence. 40S ribosomal subunit binds to the
Cap structure aided by the Cap-binding complex eIF4F. The complex along with
initiator tRNA moves along the mRNA to find the proper AUG codon (Scanning).
Bacterial Translation Initiation
Features of Eukaryotic mRNA that promote
recruitment of ribosome
Eukaryotic Translation Initiation: Scanning machanism
Eukaryotic Translation Elongation
Translation Factors
Different Initiation factors, Elongation and Termination
factors are required for protein synthesis.
Some factors provide catalytic function, some provide energy
for the movement of mRNA through the ribosome and others
stabilize the complexes.
Many translational factors bind GTP when they are active
and become inactive when bound to GDP
Prokaryotic Translation Factors:
Initiation Factors: IF-1, IF-2 and IF-3.
Elongation Factors: EF-Tu, EF-Ts and EF-G.
Termination Factors: RF1, RF2 and RF3.
Initiation factors in Prokaryotes
IF1: Binds and stabilizes 30S subunit, prevents premature binding of tRNAs to A
site
IF2: Binds fMet-tRNA to 30S subunit, binds GTP and stimulates its hydrolysis. IF1
and 2 work together
IF3: binds 30S subunit and prevents association of 50S subunit, enhances
specificity of fMet-tRNA to P site.
Elongation Factors in Prokaryotes
Ef-Tu: Binds GTP, brings elongator aa-tRNAs to A site of the 70S ribosome,
hydrolyzes GTP
EF-Ts: GTP-GDP exchange factor, regenerates active GTP-EF-Tu from GDP-EF-Tu
EF-G: Binds GTP, hydrolysis of GTP facilitates translocation of the mRNA
to the next codon from 5’ to 3’ direction.
Termination/Release factors in Prokaryotes
Release factors promote termination of translation & release of polypeptide
RF1: Catalyzes cleavage of polypeptide chain from tRNA and dissociation of
translocation complex, specific for UAA and UAG codons.
RF2: Functions like RF1, specific foe UGA and UAA codons.
RF1 and RF2 structure resembles that of tRNA and binds at the termination codons.
RF3: stimulates the function of RF1 and RF2
RRF: Ribosome recycling factor. When bound to ribosome, it recruits IF3 that
promotes dissociation of the 70S ribosome into 30S and 50S subunits.
Release Factor structure
RIBOSOME RECYCLING
In prokaryotes a factor called Ribosome Recycling Factor (RRF)
mediates dissociation of 50S and 30S subunits by recruiting EF-G
and IF3
RFs and RRF binding to ribosome
Peptidyl transferase reaction
Release factors
Protein Synthesis Inhibitors
Binds to a site in 30S subunit in
prokaryotes and inhibits aminoacyl tRNA
binding to A site.
Binds to 30S subunit and
alters the decoding centre
Binds peptidyl transferase
leading to wrong tRNA
centre and prevents
binding to ribosome.
correct binding of [Link]
to the A site & prevents
peptide bond formation
Prokaryotic & Eukaryotic inhibitor. Binds peptisyl transferase centre in large subunit, Acts
as a terminator of chain elongation by mimicking the 3’end of aminoacyl tRNA at A site and
acts as acceptor for the nascent polypeptide chain. In prokaryotes, it also increases error
rate by decreasing the selectivity of codon-anticodon base pairing.
Diphtheria Toxin
Diphtheria toxin cleaved into 2 fragments A and B. the A
fragments adds Adenosine diphosphate from NAD (Nicotinamide
Adenine dinucleotide) leading to ADP-ribosylation of eEF2, thus
inactivating elongation factor and ribosome translocation.
Ricin and -Sarcin: function as Ribonucleases and cleave a
phosphodiester bond in highly conserved loop in the ribosome large
subunit RNA resulting in poor binding of elongation factors leading to
inability of ribosome to activate of GTPase activity of the elongation
factors.
Eukaryotic Initiation Factors (eIFs): there are 10 initiation factors.
eIF1A: Assists eIF2 in promoting Met-tRNAiMet bind to 40S subunit & promotes
subunit dissociation.
eIF2: a GTPase, facilitates binding of initiator tRNA to 40S subunit
eIF2B, eIF3: Bind to 40S subunit, Promote formation of ternary complex of 40S
subunit, initiator tRNA and mRNA binding to 40S and scanning mRNA
eIF4F: Cap-binding complex
eIF4A: RNA helicase, removes secondary structures in mRNA.
eIF4B: binds mRNA and facilitates scanning of mRNA to locate AUG codon.
eIF4E: binds Cap of mRNA
eIF4G: binds to eIF4E and PABP and promotes circularization of mRNA.
eIF5B: GTPase, involved in entry of initiator tRNA, promotes dissociation of
other initiation factors from 40S subunit leading to joining of 60S subunit.
eIF6: Facilitates dissociation of 80S into 40S and 60S subunits
Elongation Factors (eEFs):
eEF1: GTP binding
eEF1, eEF1 : GTP exchange
eEF2: Ribosome translocation
Release Factors (eRFs):
eRF1: UAA, UAG and UGA recognition
No eRF2
eRF3: Stimulation of other RFs, promotes peptide cleavage
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