1 CYTOGENETICS TRANSLATION 02/10/2021

Pathway to making Protein
 Translation is the process of decoding the mRNA into a
polypeptide chain
o mRNA is decoded to form protein, which
contains specific series of amino acids
 Ribosomes read mRNA three bases or 1 codon at a
time and construct the proteins

CODONS AND ANTICODONS

 The genetic instructions being carried by the DNA

molecule are the ones being used to synthesize a
protein
o Synthesis of proteins occurs in the
cytoplasm
 During translation process, a cell reads an information
Translating the Genetic Message
that is found in mRNA. That information is used to
synthesize or build protein.
o mRNA always encode a polypeptide or chains
of amino acid rather than a whole protein
 The instruction for building a polypeptide are RNA
nucleotides (adenine, cytosine, uracil, guanine)
 Read in groups of 3 which are called Codon
o A triple code means that a sequence of 3 bases
that is needed to specify one amino acid
o This genetic code must translate the language
of the DNA which contains of 4 bases into the
common language of 20 common amino acid
 Biosynthesis requires the following material that are found in proteins
o Ribosome,  The 3 bases of an anticodon are complementary to the
 Site of protein synthesis 3 bases of a codon
o mRNA and tRNA  Example: Codon ACU, Anticodon UGA
 5’ to 3’ prime direction
 Bound to ribosome in the course of THE GENETIC CODE
protein synthesis are responsible
for the correct order of amino acid
in the polypeptide chain
Steps in Protein Biosynthesis
1.) Amino acid activation
 Involved both tRNA and amino aclt
synthease enzyme
2.) Chain initiation
3.) Elongation
4.) Termination
TRANSLATION

 Use the code by reading from the center to the outside

 Example: AUG codes for methionine
 2 CYTOGENETICS TRANSLATION 02/10/2021

o Allows fewer tRNA to cover all the codons

of the genetic while still making sure the
code is read accurately and properly.
o Wobble base of the anti-codon is the one at
the 5’ end of the tRNA. Which forms
hydrogen bonds with the 3rd base of the
mRNA is found near 3’ prime end
o Wobble pairing would help the cell that it
could invest less energy in the in the
synthesis tRNA.

Materials needed for Translation (Initiation stage)

1. Mature mRNA
2. Ribosomes
3. Initiator tRNA
4. GTP
5. eIFs
 There are 64 codons
o Sequence of 3 bases
***Charged tRNAs (needed for elongation)
o Have assigned meaning
o Only 61 are coding for amino acid
1. Mature mRNA
 Because the remaining 3 codons,
will serve as start codon or the
initiator codon
 Start codon will always bring base
sequence adenine, uracil, and
guanine that specifies methionine
 UAA, UAG, UGA are codons that
do not code for amino acid
o An amino acid can have multiple codons
o 2 amino acid such as methionine (AUG)
and tryptophan (UGG) have only one
codon each

If there are 64 codons, how can there be fewer that 64

tRNA molecules
 Some tRNA bonds to one codon exclusively but many
of them can recognize more than one codon because
of variation in the hydrogen bonding
 Atypical base pairing
o Wherein there is unusual base pairing for
nucleotides other than A-U and G-C. That
can form in the 3rd position of the codon
 Wobble pairing
o Lets the same tRNA recognize multiple
codons for the amino acid it carries
o Ensures that codons are read correctly
despite the wobble.
 START and STOP codons on mRNA are
essential for proper mRNA translation
 mRNA that has undergone modification
wherein the introns or the intervening sequence
are spliced so that what will remain in the
mRNA are the exons or coding sequence
 also undergone processing steps which are
capping at the 5’ end, addition of adenine
bases in the process called polyadenylation
o important because it makes the mRNA
stable such that in their way to the
ribosome, they won’t be degraded
easily by the enzyme
o the ends that have undergone
processing can help mRNA know in its
way to the ribosome
 mRNA is carrying genetic sequence in the form
of RNA bases which are read by ribosomes in
sequence of 3 bases called codon
o therefore, the genetic sequence by
mature mRNA that will serve as a
template to assemble a chain of amino
acid that will eventually form a protein
that is needed by the cell.
 START and STOP codons on mRNA are
essential for proper mRNA translation
3 CYTOGENETICS TRANSLATION 02/10/2021

o In Prokaryotes, can have multiple

shine -dalgarno box
2. Ribosomes
 Made of a large and small subunit
 Composed of rRNA (40%) and proteins (60%)
 Have 3 sites for tRNA attachment – E, P, A
 Ribosomes are made up of proteins and
ribosomal RNA (rRNA)
 Ribosome Binding Sites
o Not all region of the mRNA molecule o 1 mRNA binding site
corresponds to particular amino acid. o 3 tRNA binding sites
o In particular there is an are in the mRNA - E, P, A
which is near in the 5’ end that is known as  Ribosome tRNA binding sites
untranslated region UTR or leader
sequence.
o UTR – located between first
nucleotide that is transcribed and
the start codon
o The untranslated region does not
affect the amino acid sequence.
o Even if it doesn’t specify a specific
amino acid it contains a ribosome
binding site.
 Is composed of series of
sequence of nucleotides
 Where the actual ribosome
binding happens.
 Prokaryotic cell ribosome
binding site is called Shine-
Dalgarno box (named after
John Shine and Lynn
Dalgarno) o A site
 AGGAGG - Aminoacyl-tRNA site
sequence (binding - Holds the tRNA carrying the next
site for ribosome) amino acid to be added
 Also found in o P site
eukaryotes - Peptidyl-tRNA site
 Vertebrates which - Holds the tRNA molecule carrying the
characterized by Kozak growing polypeptide chain
box (Marilynn Kozak) o E site
 The 5’ UTR for prokaryote - Exit site
is shorter - Where tRNA molecules leave the
 Median length for humans ribosome
is 117 nucleotides of the 5’
UTR
 When leader sequence is Transfer RNA (tRNA)
long then it may contain
regulatory sequence like
enhance sequence and
etc.
o Shine-Dalgarno Sequence
 5’ untranslated region
where sight of ribosome
binding is
 Purine rich sequence
 Able to distinguish initiator
start codon from that of the
internal codon AUG
- Single stranded molecule with attachment
 AUG nearest to the 5’ end
site at one end for an amino acid
of the mRNA is the one
- Opposite end has three nucleotide bases
selected is the start
called anticodon
 4 CYTOGENETICS
 Roles of tRNA
o Serve as molecular bridges
 Connect mRNA codon to the amino
acid that it encodes through the
anticodon
 Each tRNA reads one or few
codons and brings the specific
amino acids matching this codon.
 Should be charged so that the
appropriate amino acid sequence
attached to it on the other end and
be delivered to the A site of the
ribosome (uncharged tRNA are
recycled; charged tRNA is aided by
enzyme Aminoacyl-tRNA
synthetase)
tRNA activation (charging) by aminoacyl tRNA
synthetases
 Two important functions
o Implement genetic code
o Activate amino acids for peptide bond
formation
 Key enzyme
o Aminoacyl-tRNA synthetases
Amino Acylation process – attachment of amino acid to a
tRNA with the help of aminoacyl-tRNA synthetase
TRANSLATION 02/10/2021
STEPS (2-step process)
 (1) ACTIVATION: amino acid + ATP  aminoacyl-
AMP + PPi
 Amino acid and ATP needed for the step must
enter the active site of the enzyme
 Once bound to the active site, then the adeno
monophosphate is joined to the amino acid
 Adenosine monophosphate will join the amino
acid with the help of an amino acid that triggers
the release of pyrophosphate
 (2) CHARGING: aminoacyl-AMP + tRNA 
aminoacyl-tRNA + AMP
 Uncharged tRNA displaces AMP once attached
to the active binding site of the enzyme and
once bound to the amino acid
 tRNA will bind to the site emptied by AMP
 tRNA replaces the empty site
 tRNA binds itself to the amino acid = charged
tRNA or aminoacyl Trna
 charged tRNA will then be released from the
site of the enzyme
 active site is now available for another binding
Two-stage reaction
 In the first stage, the amino acid is activated with ATP
 In the second stage of the reaction, the amino acid is
attached with a high-energy bond to the ribose sugar at
the 3’ end of the transfer RNA
 Note: There are 20 different aminoacyl-tRNA
synthetase for the 20 different amino acids
Charged tRNA
 Amino acid attached by carboxyl group to ribose of last
ribonucleotide of tRNA
 Last 3 nucleotides of tRNAs are –CCA-3’
 Cytosine nucleotide and adenine nucleotide
 Cytosine – cytosine – adenine
 Carboxyl group of the amino acid specifically binds to
the ribose of the last ribonucleotide of the Trna
 Adenine – last nucleotide among the three (where the
carboxyl group of the amino acid binds)
Translation has 3 phases:
1. Initiation
2. Elongation
3. Termination
 5 CYTOGENETICS
EUKARYOTIC TRANSLATION
 Initiation of Translation
 Where the difference from prokaryotes and
eukaryotes lies
- In eukaryotes bind to the 5’ cap and
moves down to the mRNA until they
reach the first AUG base sequence
(which is the codon that codes the
initiator – amino acid methionine)
- Ribosomes do the scanning (from 5’
end of mRNA)
 Initiation complex signals the start of translation
process
 Ribosomes bind to the 5’ cap, then move down the
mRNA until they reach the first AUG, the codon for
methionine. Translation starts from this.
 Note that translation does not start at the first base
of the mRNA. There is an untranslated region (5’
UTR)
 An initiation codon marks the start of an mRNA
message
 AUG – methionine
 Start of mRNA message is found where
initiation codon is located
TRANSLATION 02/10/2021
Step 1 – Initiation
 mRNA transcript start codon AUG attaches to the small
ribosomal subunit
 the initiation process involves first joining the mRNA,
the initiation methionine tRNA, and the small ribosomal
subunit
 The large ribosomal subunit then joins the complex
 An initiation codon marks the start of an mRNA
message
 mRNA, a specific tRNA, and the ribosome subunits
assemble during intiation
 The process:
1. Initiator tRNA (Met-tRNA) gets loaded onto the
small ribosomal subunit (40s)
2. Eukaryotic initiation factors (eIFs) gets loaded as
well on the 40s ribosomal subunit
3. A pre-initiation complex is formed containing (40S –
Met-tRNA – eIFs).
4. The complex (40S – Met-tRNA – eIFs) binds to the
5’ end of the mRNA (5’ Cap).
5. More eIFs are bound to the 5’ Cap
 6 CYTOGENETICS
6. The complex (40S – Met-tRNA – eIFs) moves
along the transcript (5’ -3’) in search for the first
AUG
7. The process is called “scanning” of mRNA
8. When first AUG is found, EIFs dissociate
- Detached from the pre-initiation
complex
9. The large ribosomal subunit (60S) assembles to the
complex at the start codon
- eIF6 assembles to the complex at the
start codon (binds to the ribosomes)
10. The initiator tRNA is positioned in the P site of the
ribosome and the A site (adjacent to P site) is
available to receive to next charged tRNA (next
tRNA bearing the amino acid)
- Forms translation initiation complex
(small ribosomal unit, mRNA, large
ribosomal unit, and tRNA seated at the
p site of the ribosomal unit)
- Large ribosomal unit is bound with eIF6
= because it prevents the large
ribosomal subunit from reassociating
with small subunits and so its removal
is required first
TRANSLATION 02/10/2021
 Remember! Chain initiation is part of the eukaryotic
initiation
 STEP 1: Assemble 43s pre-initiation complex,
aside from that, tRNA is bearing the initial amino
acid, Methionine, which is attached to a specialty
RNA that serves as the initiator tRNA
 In eukaryotes there is no formulation of the
methionine (in prokaryotes, formulation of
methionine is recognized)
 Met tRNA is delivered to the 40s ribosomal unit as
a complex with guanosine triphosphate and
transcription factor eIF2
 40s ribosome is also bound to other initiation
factors (3 and 1A) – different from prokaryotes (1st
tRNA binds to a ribosome without the presence of
mRNA)
 STEP 2: mRNA is recruited
 Note: In the eukaryotic mRNA, there is no shine-
delgarno sequence for the location of the start
codon, instead it’s the Kozak Sequence
 Kozak Sequence – characterized by a consensus
sequence of ACCAUGG
 5’ cap of mRNA orients the ribosome to the correct
codon via the process of scanning (driven by ATP
hydrolysis)
 mRNA has a prior binding with the eIF 4e, 4a, 4g
before it binds to the pre initiation complex
 Remember! eIF 4e is a cap-binding protein that
forms complex with several other eukaryotic
initiation factors
 43s is believed to be recruited to a 5’ cap structure
at the end of mRNA via pre-existing mRNA that
already is in complex with 4e, 4g and 4a
- Recruited to the pre-existing mRNA
 Poly A binding protein (Pab1p) – essential because
it links poly A tail to the eIF 4g forming a loop
 7 CYTOGENETICS
 eIF 4G serves as a multi-purpose adaptor to
engage the methyl guanine cap, 4E complex
 loop – promotes both translation and stability of
Mrna
 eIF40s complex that was initially positioned
upstream of the start codon
 by then it starts to move downstream from 5’ – 3’ to
scan for the initation codon
 until it encounters first AUG base sequence
 with that, then the eukaryotic initiation factor
dissociate from the pre initiation complex
 Large ribosomal subunit starts to bind with the
small ribosomal unit with the tRNA
 Dissociation of initiation factors from pre-initiation
complex needs GTP hydrolysis
 The 80s complex/ translation initiation complex
cues that translation elongation will commence,
 Elongation (in Eukaryotes)
 The peptide chain in eukaryotes is very similar to
prokaryotes (but structure of eukaryotic ribosome is
different. No E site)
 They also differ in elongation factors
o Eukaryotic organism: elongation comes in 2
forms
1. eEF1 (eEF1A & eEF1B)
2. eEF2
o Prokaryotes:
1. eEF1A = EF-Tu in prokaryotes
2. eEF1B = EF-Ts
3. eEF2 = EF-G
- ribosome moves along mRNA in 5’ – 3’
require EF-G, in a process called
translocation
 tRNA that corresponds to the 2nd codon of mRNA
can then bind to the A site of the large ribosomal
subunit
 tRNA bearing the next amino acid which
corresponds to the next codon in the mRNA
(proofread by ribosomes)
TRANSLATION 02/10/2021
 Binding of tRNA to the second codon requires
energy source – GTP
 upon binding of tRNA amino acid complex to the A
site, the GTP is cleaved to form the guanosine
diphosphate and relased along with EF and is
recycled by EF-TU (prokaryote) and EF1A
(eukaryote)
 Next step: peptide bonds formation – between the
adjacent precious amino acid and newly added
amino acid
 Bond is formed through a peptidyl transferase
activity
 For many years, it was thought that an enzyme
catalysed this step. Recent evidence indicate that
the transferase activity is actually a catalytic
function of the rRNA
- 40% of ribosomes is consist of rRNA
 As peptide bond is formed the ribosome shifts and
moves three bases (reads 4 bases at a time
towards 3’ end)
- Another amino acid is being carried by
a new tRNA in the a site
- Once it reaches at the a site, another
peptide bond is formed by rRNA
- until It encounters stop codon
 Ribosome shift from one codon to another towards
3’ end is called translocation process.
 Elongation factors functions:
o Eukaryotic organism: elongation comes in 2
forms
1. eEF1 (eEF1A & eEF1B) – particularly 1A,
responsible for enzymatic deliver of
aminoacyl tRNA to the ribosomes
- 1B: nucleotide exchange factor that is
required to regenerate elongation
factor 1A from its inactive form to active
form
2. eEF2 – responsible for translocation of
peptidyl tRNA from the A site to the P site
- making a site vacant for the next
aminoacyl tRNA to bind
 2nd codon can now bind to the A site which require
elongation factors
 Stage where amino acids are being added in the
growing polypeptide chain (peptidyl site – where
tRNA that is carrying the growing peptide chain is
being held)
 mRNA is read one codon at a time (triplets of bases
at a time)
 Amino acid matching it codon is added to the
growing polypeptide chain (ribosomes does
proofreading to the mRNA's genetic information)
 STEP 1: Codon recognition
o Incoming aatRNA to A site
o H bonds form between the mRNA codon and
tRNA anticodon
o Energy is required
 The mRNA moves a codon at a time relative to the
ribosome
 8 CYTOGENETICS TRANSLATION 02/10/2021

o a tRNA pairs with each codon, adding an amino
acid to the growing polypeptide
o a STOP codon causes the mRNA-ribosome
complex to fallapart
 STEP 2: Peptide bond formation
o Ribosome catalyzes the formation of a peptide
bond
 Between the amino acid in the p-site to the
amino acid in the a-site
 Involves the carboxyl end of the
polypeptide chain
o Result:
 Polypeptide chain is longer by one amino
acid
 Polypeptide chain is transferred to tRNA at
the A site
 STEP 3: Translocation
o Translocation requires energy
o Ribosome moves:
 tRNA from P site to E site: leaves ribosome
 tRNA from A site to P site: polypeptide
returns to P site, ready for next
polymerization
o A site is now empty
 Next aatRNA can bind
polypeptide to separate. The new polypeptide is
completed.
 At stop codon a protein called release factor binds to A
site (no tRNA for stop codon, thus no aatRNA)
 Release factor:
 Adds of water molecule instead of amino acid to
polypeptide
 Polypeptide hydrolysed from tRNA in P site
released
 Translation complex disassembles
End product – PROTEIN!
 The end products of protein synthesis is a primary
structure of a protein
 A sequence of amino acid bonded together by peptide
bonds
Post-translational Modification
 These modifications give the proteins specific functions
and target the proteins to specific areas within the cell
and the whole organism
- Protein folding to achieve functional
proteins needed by cell

 Termination
Termination in eukaryotes is similar to that in prokaryote
 Finished polypeptide chain is being released
 The ribosomal complex dissociates
 Eukaryotic release factor (eRF1) recognizes all stop
codons
 Prokaryotes:
1. RF1 - recognizes stop codons UAG and UAA
2. RF2 – recognizes stop codons UGA and UAA
3. RF3 – recycling role in translation by facilitating
removal of RF1 and RF2 from ribosome
following peptide release; quality control
 Other release factors help in termination and
disassembling of the
ribosome complex.

 Three codons are called “stop codons”. They code for

no amino acid, and all protein-coding regions end in a
stop codon.
 When the ribosome reaches a stop codon, there is no
tRNA that binds to it.
 Instead, proteins called “release factors” bind, and
cause the ribosome, the mRNA, and the new
 Leader sequences at their n-terminal ends – directing
protein to their proper destination
 Recognized and removed by proteases in the
endoplasmic reticulum
 9 CYTOGENETICS TRANSLATION 02/10/2021

 Finish protein enters golgi apparatus directing it to its

final destination
 Protein undergoes protein processing to ensure that its
functional

PROKARYOTIC TRANSLATION
 Prokaryotic Ribosome – mRNA recognition
 16S rRNA binds to an mRNA at the ribosomal-
binding site or Shine-Dalgarno box
 One codon for methionine, but Marcker & Sanger find 2
tRNAs, attached to either :
 tRNAs have: different base sequences and same
anticodon
 Initiation: IF3 keeps 50S subunit from binding: IF1
enables 30S subunit to bind mRNA via H-bonding to
16S rRNA:
 Cytosol fractionation and reconstitution reveal:
 Completion of Ribosome assembly
 Elongation – 1: entry of aa2-tRNAaa2 requires GTP and
elongation factors (EFs)
1. Two EFs participate to use and the provide GTP
free energy
2. Ribosome ‘sites’:
o A = amino acid entry site on ribosome;
o P = peptidyl site;
o E = exit site
 Elongation – 2: Peptide bond formation
 Peptidyl transferase (rRNA ribozyme) catalyzes
condensation reaction to form peptide linkage
between fmet and the second encoded amino acid
in the A site
 Elongation – 3: translocation along mRNA, catalysed
by translocate enzyme on ribosome
 Note that one full cycle of elongation has cost 3
NTPs!
o To make aa-tRNA,
o To bring each aa-tRNA to A site
o To move ribosome on mRNA
 Termination: ribosome stalls at stop codons: (TAA,
TAG, TGA in the gene; UAA, UAG or UGA in the
mRNA)
1. Release factor enters A site, GTP is hydrolysed
2. Polypeptide is hydrolysed from last tRNA and
released from ribosome