LICYAYO, Michel Paulette

Chem 2065 AY2022-23

Chapter 12 Protein Synthesis: Translation of the Genetic Message

The overall objective of this chapter is to explain how information in the mRNA
is translated into a functional protein
In greater detail, you should be able to:
1. Provide an outline of the translation process
● Activation of the amino acid – Before an amino acid is incorporated into a
growing chain, it must first be activated. This involves both the tRNA and an
enzyme called aminoacyl-tRNA synthetases. Amino acid is covalently bonded to
the tRNA in the process forming the aminoacyl-tRNA.
● Chain initiation – first step in the actual formation of the polypeptide chain. The
first aminoacyl-tRNA is bound to the mRNA at the site that encodes the start of
polypeptide synthesis. In this complex, mRNA and ribosomes are bound to each
other. The next aminoacyl-tRNA forms a complex with the ribosome and with
mRNA.
● Chain elongation – A peptide bond is formed between the amino acids in this
step. This process repeats itself until the polypeptide chain is complete
● Chain termination - newly formed protein is released from the ribosome
2. Explain the features of the genetic code (codon, triplet, nonoverlapping,
commaless, unique, degenerate, universal with some exceptions)
 The genetic message is contained in a triplet, nonoverlapping,
commaless, degenerate, universal code.
o Triplet code – a sequence of three bases, called the codon, is
needed to specify one amino acid in a protein
o Nonoverlapping – no bases are shared between consecutive
codons; ribosome moves along the mRNA three bases at a
time rather than one or two at a time
o Commaless – no intervening bases exist between codons
o Degenerate – more than one triplet can encode the same
amino acid. This acts as a buffer against deleterious mutations.
o Universal code – same in all organisms

3. Explain how the genetic code was determined (use of synthetic mRNA, filter-
binding assay)
● Synthetic mRNA – synthetic polynucleotides used as messengers. When the
homopolynucleotides are used as synthetic mRNA for polypeptide synthesis, it
produces homopolypeptides. This procedure is used to establish the code for the
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

four possible homopolymers quickly. When an alternating copolymer is the

messenger, an alternating polypeptide is the product.
● Filter-binding Assay – In this technique various tRNA molecules are mixed with
ribosomes and synthetic trinucleotides that are bound to filter. The mixture of
tRNA is passed through the filter and when a radioactive label is detected, then it
is known that the particular tRNA binds. If the radioactive label is in the solution
that flowed through the filter, then the tRNA did not bind.
4. Cite the significance of the following codons: AUG, UAA, UAG, UGA, UGG
● AUG – a start codon; codes for methionine
● UAA, UAG, UGA – stop codons which encodes a release factor that causes
translation to cease
● UGG – the only codon that codes for tryptophan
5. Describe the nature of the codon-anticodon pairing between the mRNA and
the tRNA
When an amino acid is incorporated during the protein synthesis, a codon forms
with a complementary anticodon of a tRNA. An anticodon is a sequence of three
base pairs in tRNA that hydrogen bonds with the mRNA triplet that specifies a
given amino acid
6. Explain the nature and the advantage of the wobble in the third base pairing
between mRNA and the corresponding tRNA in eliminating the need of having all
64 tRNA molecules present in the cell
● A wobble is the possible variation in the third base of a codon allowed by
several acceptable forms of base pairing between mRNA and tRNA. It applies to
the first base of an anticodon (on the 5’ end), but not the second or third base.
The first base of the anticodon hydrogen bonds to the third base of the codon,
the one at the 3’ end.
● The wobble model provides understanding into some aspects of the
degeneracy of the code. In many cases, the degenerate codons for a given
amino acid differ in the third base, the one that pairs with the wobble base of the
anticodon. Fewer different tRNAs are needed because a given tRNA can base-
pair with several codons then a cell would have to invest less energy in the
synthesis of needed tRNAs. The wobble also minimizes the damage caused by
misreading of the code.
7. Explain the need for activating amino acids to form a peptide bond (Recall your
organic chemistry)
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

Activating the amino acids is required for translation to occur. It ensures that the
correct amino acid will be recognized and that there is sufficient energy for the
formation of the peptide bond.
8. Describe the steps in activating amino acids by the enzyme aminoacyl-tRNA
synthetase (energy requirements, mixed anhydride, phosphoester bond, why is
there only one ATP involved--yet it is counted as if two ATPs are involved, Class
I and Class II aminoacyl-tRNA synthetases)
Step 1: Amino acid forms a covalent bond to an adenine nucleotide, producing
an aminoacyl-AMP. Free energy of hydrolysis of ATP provides energy for bond
formation. The aminoacyl moiety is transferred to the tRNA, forming the
aminoacyltRNA.
Step 2: An ester linkage is formed between amino acid and either the 3’-hydroxyl
or the 2’-hydroxyl of the ribose at the 3’ end of the RNA. Two classes of
aminoacyltRNA synthetases: (a) Class I loads amino acid onto 2’ hydroxyl while
(b) Class II uses the 3’ hydroxyl.
9. Explain why the selectivity of the aminoacyl-tRNA synthetase is sometimes
referred to as the "second genetic code" (activity of the aminoacyl-tRNA
synthetase, sites of selective recognition for various amino acids, position 73,
acceptor stem, variable pocket, variable loop, anticodon, variable stem-loop)
 Two-stage reaction allows for selectivity to operate at two levels: amino
acid and tRNA.
o The fact that aminoacyl-AMP is bound to an enzyme is used in the
specificity of the first stage.
 Selectivity resides in the tRNa not in the amino acid
o The selective recognition of tRNAs by aminoacyl-tRNA synthetases
is the one depended on by the second aspect of selectivity.
 Specific binding sites on tRNA are recognized by aminoacyl-
tRNA synthetases
 Exact position of the recognition site varies with different
synthetases which is a source of greater specificity.
 Anticodon is not always part of the tRNA that is recognized
by the aminoacyl-tRNA synthetase
 Recognition of correct tRNA is vital to the fidelity of
translation because final proofreading mostly occur in this
step
10. Differentiate between the components of the ribosome
● Ribosomes have specific architecture which facilitates the binding. During
protein synthesis. A tRNA molecule (orange) is base pairing with part of the
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

mRNA (gold) on the left. The tRNA extends to the peptidyl transferase center on
the right.
● Ribosomes consists of small and large subunits. The large sits on top of the
small subunit, and between is the RNA template. The small subunit decodes
genetic message while the large catalyzes peptide bond formation.

11. Describe the formation of the initiation complex (Shine-Dalgarno sequence,

IF-1, IF-2, IF-3, fmet-tRNA-fmet(superscript), 30S initiation complex, 70S
initiation complex, fmet vs met)
 The initiation complex formation is required in the start of the polypeptide
synthesis. The 30S ribosomal subunit binds to mRNA and fmet-tRNAfmet
with the presence of GTP and three initiation factors (IF-1, IF-2, IF-3),
forming the 30S initiation complex. The 50S ribosomal subunit is added,
forming the 70S initiation complex.
o IF-3 protein facilitates binding of mRNA to the 30S ribosomal
subunit; prevent premature binding of the 50S subunit
o IF-2 binds GTP and aids in the selection of the initiator tRNA from
all other aminoacylated tRNAs.
o IF-1 appears to bind to IF-3 and to IF-2 and facilitates the action of
both. It also catalyzes separation of the 30S and 50S ribosomal
subunits being recycled for another round of translation.
 30S initiation complex is the resulting combination of mRNA, 30S
ribosomal subunit, and fmet-tRNAfmet.
 70S initiation complex is produced when a 50S ribosomal subunit binds to
the 30S initiation complex.
12. Explain how the leader segment Shine-Dalgarno sequence plays a role in
guiding the correct binding of the mRNA to the 30S ribosomal subunit
purine-rich leader segment called the Shine-Dalgarno sequence (5’-GGAGGU3’)
precedes the start signal. This sequence usually lies about 10 nucleotides
upstream of initiation codon and acts as a ribosomal binding site. This sequence
on the 16S ribosomal RNA part of the 30S subunit and aligns it for proper
translation beginning with the AUG start codon.
13. Describe the nature of the P (peptidyl) site, A (aminoacyl) site, and E (exit)
site in the 70S ribosome
● P (peptidyl) – binds a tRNA carrying a peptide chain; one initially occupied by
the fmet-tRNAfmet in the 70S initiation complex.
● A (aminoacyl) – binds an incoming aminoacyl-tRNA; site where the second
aminoacyl-tRNA binds
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

● E (exit) – carries an uncharged tRNA that is to be released from the ribosome

14. Describe the process of chain elongation (entry of correct aminoacyl-tRNA
based on the correct codon with the aid of EF-Tu*GTP and its subsequent
recycling with the aid of EF-Ts, formation of the peptide bond, translocation
of the mRNA and tRNA relative to each other with the aid of EF-G*GTP, exit
of the uncharged tRNA, entry of the next aminoacyl-tRNA based on the
correct codon)
● Step 1: Aminoacyl-tRNA is bound to the A site on the ribosome; this requires
the elongation factor (EF-Tu (Tu) and the GTP; the P site on the ribosome is
already occupied ●
Step 2: Elongation factor EF-Tu is released from the ribosome and regenerated
in a process requiring elongation factor EF-Ts (Ts) and GTP
● Step 3: The peptide bond is formed, leaving an uncharged tRNA at the P site
● Step 4: The uncharged tRNA is released in the translocation step. The
peptidyl-tRNA is translocated to the P site, leaving an empty A site. The
uncharged tRNA is translocated to the E site and subsequently released.
Elongation factors EF-G and GTP are required
15. Explain why the selectivity of EF-Tu is important to E. coli (and presumably other
prokaryotes)
EF-Tu is involved in another level of translation fidelity. EF-TU is efficient at
delivering activated tRNA to the ribosome when the correct amino acid is bound
to the correct tRNA. If it is mismatched, then either the EF-Tu does not bind the
activated tRNA very well, which results to not delivering it well to the ribosome, or
it binds the activated tRNA too well in which it does not release it from the
ribosome.
16. Explain the nature of the peptidyl transferase (RNA as enzyme, ribozyme)
Peptidyl transferase catalyzes a reaction that forms the peptide bonds.
17. Explain how inhibitors like puromycin helped elucidate the mechanisms involved
in translation (Assignment: read on other antibiotics and their modes of
inhibition in the translation process)
Puromycin is a structural analog for the 3’ end of the aminoacyl-tRNA, making it
useful in studying chain elongation. This inhibitor binds to the A site and a
peptide bond is formed between the C-terminus of the growing polypeptide and
the puromycin. Premature and defective protein results from the peptidyl
puromycin weakly bound to the ribosome and dissociates easily. The puromycin
also binds to the P site and blocks the translocation process. Existence of the A
and P sites were determined by these experiments with puromycin.
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

18. Explain how protein synthesis is terminated (chain termination, UAA, UAG,
UGA, RF-1, RF-2, RF-3)
 To terminate protein synthesis, there has to be a stop signal. The codons
UAA, UAG, UGA are the stop signals. These codons are recognized by
the release factors. One of the two protein release factors, RF-1 or RF-2,
is required as is GTP, which is bound to a third release factor, RF-3.
o RF-1 – binds to the UAA and UAG
o RF-2 – binds to UAA and UGA
o RF-3 – does not bind to any codon but facilitate the activity of the
other two release factors
 Release factors do not only block the binding of a new aminoacyl-tRNA
but it also affects the activity of the peptidyl transferase so that the bond
between the carboxyl end of the peptide and the tRNA is hydrolyzed;
GTP is hydrolyzed in the process; the whole complex dissociates, which
sets free the release factors, tRNA, mRNA, and the 30S and 50S
ribosomal subunits
19. Explain why selenocysteine (Sec) is classified as the 21st amino acid and yet is
not part of the 20 common amino acids (codon UGA, specialized elongation
factors [SelB and EFsec])
● Selenocysteine (Sec) is the only amino acid that both lacks its own tRNA
synthetase and is synthesized while bound to its tRNA. It has been found in the
active sites of enzymes involved in clearing reactive oxidative molecules during
thyroid hormone activation.
● The codon for Sec is UGA which is normally a translation stop codon. During
translation of an mRNA which should lead to a Sec-bearing protein, the UGA is
interpreted differently due to interactions with specialized elongation factors, SelB
in bacteria and Efsec in humans.
20. Explain how prokaryotes are able to synthesize multiple copies of a protein
simultaneously (polysomes, coupled translation)
In prokaryotes, translation begins right after mRNA transcription. It is possible for
the mRNA molecule being transcribed to have a number of ribosomes attached
to it that are in various stages of translating that mRNA. It is also possible for
DNA to be in the various stages of being transcribed. In this situation, several
RNA polymerase molecules are attached to a single gene producing several
mRNA molecules with each has number of ribosomes attached to it. This is
possible in prokaryotes, the coupled translation, because of the lack of
compartmentalization.
21. Provide an overview of eukaryotic transcription [As with replication and
transcription, eukaryotic translation has the same general picture as prokaryotic
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

translation but with, more factors involved]. Just take note of the more prominent
differences not all the differences. IF vs eIF, 40S subunit, 43S preinitiation
complex, 80S initiation complex, Kozak sequence, mRNA looping)
 Chain initiation process is the most different among the eukaryotes and
prokaryotes. Thirteen more initiation factors are given the designation eIF,
or the eukaryotic initiation factor.
o Step1: assembly of the 43S preinitiation complex ▪ Methionine is
usually the initial amino acid; attached to a special tRNA that
serves as the initiator tRNA
 NO fmet in eukaryotes
 Met-tRNA is delivered to the 40S ribosomal subunit as
complex with GTP and eIF2
 40S ribosome also bound to eIF1A and eIF3
o Step2: mRNA is recruited
 No Shine-Dalgarno sequence for the location of start codon
 5’ cap orients the ribosome to the correct AUG via scanning
mechanism driven by ATP hydrolysis
 Poly A binding protein (Pab 1 p) links the poly A tail to
eIF4G; the eIF-40S complex is initially positioned upstream
of the start codon and moves downstream until it encounters
the first AUG in the correct context
 This context is determined by few bases surrounding the
start codon called the Kozak sequence
o Step3: 60S ribosome is recruited forming the 80S initiation
complex
 GTP is hydrolyzed and initiation factors are released
 Chain elongation in eukaryotes is similar to prokaryotes. However, the
structure of the eukaryotic ribosome is different because there is no E site,
only the A and P sites.
o Two eukaryotic elongation factors: eEF1 and eEF2. ▪ eEF1 –
consists of eEF1A, counterpart of EF-Tu in prokaryotes, and
eEF1B, equivalent of EF-Ts in prokaryotes
 eEF2 – counterpart of EF-G in prokaryotes causing
translocation
 Chain Termination in eukaryotes only has one release factor that binds to
all three stop codons and catalyzes the hydrolysis of the bond between
the C-terminal amino acid and the tRNAs
22. Explain how suppressor tRNAs can allow translation to continue (Suppressor
tRNAs are modified forms of tRNA that correspond to the 'stop codons' if they
appear too early in the translation process --- perhaps due to mutation or error by
the RNA polymerase)
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

Suppressor tRNA is a special tRNA that allows translation to continue through a

stop codon. They tend to be found in cells where a mutation has introduced a
stop codon. It is a tRNA with usually a mutation in the anticodon that recognizes
a stop codon and inserts an amino acid in its place.
23. [Ponder on the possibility of coupled transcription and translation in eukaryotes]
The start codon is not AUG. The mammalian immune system produces peptides
with the objective of displaying them on the surface of major histocompatibility
complexes. CUG, rather not AUG, is commonly utilized as the start codon in
peptide synthesis, according to research. This binds to tRNA-Leu, leaving leucine
as the N-terminal amino acid.
24. [Ponder on the dangers of believing everything you are told from the evidence
that CUG is used as a start codon in mammalian immune system, and that this
start codon binds to tRNA-Leu]
Some amino acid residues are also covalently modified, as in the conversion of
proline to hydroxyproline. Other covalent modifications can take place, an
example being the addition of carbohydrates or lipids to yield an active final form
of the protein in question. Proteins can also be methylated, phosphorylated,
arginylated, and ubiquitinylated.
25. Describe the posttranslational modification of proteins (cleavage of leader
sequences, endoplasmic reticulum, Golgi apparatus, breaking of peptide bonds,
methylation, hydroxylation, phosphorylation, formation of disulfide linkages,
addition of prosthetic groups, arginylation, ubiquitinylation, etc.)
 N-formylmethionine is cleaved off in prokaryotes. Precursor specific
bonds can be hydrolyzed; proteins destined for export to specific parts
have leader sequences at their N-terminal ends which direct proteins to
their proper destination, are recognized and removed by proteases
associated with the endoplasmic reticulum.
 Finished protein enters the Golgi apparatus which directs final destination
 Other substances can be linked to the newly formed polypeptide o
Cofactors are added and disulfide bonds are formed
 Amino acid residues are modified covalently
 Proteins can also be methylated, phosphorylated, arginylated, and
ubiquitinylated
26. Describe the roles of chaperones and the ribosome itself in preventing
protein misfolding
 Some proteins may form aggregates with other proteins or fold with
incorrect secondary and tertiary structures unless they interact first with a
chaperone.
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

 The primary structure of a protein determines the three-dimensional

structure but often they also need help from chaperones to arrive at the
correct structure. Presence of ribosome is able to confer the ability of the
protein to fold correctly, acting as its own chaperone to prevent protein
misfolding.
27. Explain how proteins are degraded (proteasomes, lysosomes,
ubiquitinylation)
 Degradation pathways are restricted to degradative subcellular organelles
such as lysosomes, or to macromolecule structures called proteasomes.
Via specific signal sequences, proteins are directed to lysosomes. One the
protein is in the lysosome, destruction is nonspecific.
 In eukaryotes, ubiquitinylation is the most common mechanism for
targeting protein for destruction in the proteasome. When ubiquitin is
linked to a protein, it condemns that protein to destruction in a
proteasome.