Protein synthesis

Dario Leonardo Balacco, PhD

School of Dentistry
University of Birmingham, UK
d.l.balacco@bham.ac.uk

3.4.2021
The central dogma of biology

transcription
translation

reverse
transcription

DNA RNA Protein

From transcriptome to proteome

ACATTCAAGAGGAGCTTTCAGGCGATCTGGAGAAAGAACGGCAGAACACACAGCAAGGAAAGGTCCTTTC
TGGGGATCACCCCATTGGCTGAAGATGAGACCATTCTTCCTCTTGTGTTTTGCCCTGCCTGGCCTCCTGC
ATGCCCAACAAGCCTGCTCCCGTGGGGCCTGCTATCCACCTGTTGGGGACCTGCTTGTTGGGAGGACCCG
GTTTCTCCGAGCTTCATCTACCTGTGGACTGACCAAGCCTGAGACCTACTGCACCCAGTATGGCGAGTGG
CAGATGAAATGCTGCAAGTGTGACTCCAGGCAGCCTCACAACTACTACAGTCACCGAGTAGAGAATGTGG
CTTCATCCTCCGGCCCCATGCGCTGGTGGCAGTCACAGAATGATGTGAACCCTGTCTCTCTGCAGCTGGA
CCTGGACAGGAGATTCCAGCTTCAAGAAGTCATGATGGAGTTCCAGGGGCCCATGCCCGCCGGCATGCTG
ATTGAGCGCTCCTCAGACTTCGGTAAGACCTGGCGAGTGTACCAGTACCTGGCTGCCGACTGCACCTCCA
CCTTCCCTCGGGTCCGCCAGGGTCGGCCTCAGAGCTGGCAGGATGTTCGGTGCCAGTCCCTGCCTCAGAG
GCCTAATGCACGCCTAAATGGGGGGAAGGTCCAACTTAACCTTATGGATTTAGTGTCTGGGATTCCAGCA
ACTCAAAGTCAAAAAATTCAAGAGGTGGGGGAGATCACAAACTTGAGAGTCAATTTCACCAGGCTGGCCC
CTGTGCCCCAAAGGGGCTACCACCCTCCCAGCGCCTACTATGCTGTGTCCCAGCTCCGTCTGCAGGGGAG
CTGCTTCTGTCACGGCCATGCTGATCGCTGCGCACCCAAGCCTGGGGCCTCTGCAGGCCCCTCCACCGCT
GTGCAGGTCCACGATGTCTGTGTCTGCCAGCACAACACTGCCGGCCCAAATTGTGAGCGCTGTGCACCCT
TCTACAACAACCGGCCCTGGAGACCGGCGGAGGGCCAGGACGCCCATGAATGCCAAAGGTGCGACTGCAA
TGGGCACTCAGAGACATGTCACTTTGACCCCGCTGTGTTTGCCGCCAGCCAGGGGGCATATGGAGGTGTG
TGTGACAATTGCCGGGACCACACCGAAGGCAAGAACTGTGAGCGGTGTCAGCTGCACTATTTCCGGAACC
GGCGCCCGGGAGCTTCCATTCAGGAGACCTGCATCTCCTGCGAGTGTGATCCGGATGGGGCAGTGCCAGG
From transcriptome to proteome

• Link between transcriptome and proteome.

ACATTCAAGAGGAGCTTTCAGGCGATCTGGAGAAAGAACGGCAGAACACACAGCAAGGAAAGGTCCTTTC
TGGGGATCACCCCATTGGCTGAAGATGAGACCATTCTTCCTCTTGTGTTTTGCCCTGCCTGGCCTCCTGC • The combination of 4 nucleotides should
ATGCCCAACAAGCCTGCTCCCGTGGGGCCTGCTATCCACCTGTTGGGGACCTGCTTGTTGGGAGGACCCG
GTTTCTCCGAGCTTCATCTACCTGTGGACTGACCAAGCCTGAGACCTACTGCACCCAGTATGGCGAGTGG account for all 20 amino acids.
CAGATGAAATGCTGCAAGTGTGACTCCAGGCAGCCTCACAACTACTACAGTCACCGAGTAGAGAATGTGG
CTTCATCCTCCGGCCCCATGCGCTGGTGGCAGTCACAGAATGATGTGAACCCTGTCTCTCTGCAGCTGGA
• A two-letter code would have only 42=16
CCTGGACAGGAGATTCCAGCTTCAAGAAGTCATGATGGAGTTCCAGGGGCCCATGCCCGCCGGCATGCTG
ATTGAGCGCTCCTCAGACTTCGGTAAGACCTGGCGAGTGTACCAGTACCTGGCTGCCGACTGCACCTCCA
codons.
CCTTCCCTCGGGTCCGCCAGGGTCGGCCTCAGAGCTGGCAGGATGTTCGGTGCCAGTCCCTGCCTCAGAG • A three-letter code would give 43=64 codons.
GCCTAATGCACGCCTAAATGGGGGGAAGGTCCAACTTAACCTTATGGATTTAGTGTCTGGGATTCCAGCA
ACTCAAAGTCAAAAAATTCAAGAGGTGGGGGAGATCACAAACTTGAGAGTCAATTTCACCAGGCTGGCCC • The genetic code was worked out in the 1960s.
CTGTGCCCCAAAGGGGCTACCACCCTCCCAGCGCCTACTATGCTGTGTCCCAGCTCCGTCTGCAGGGGAG
CTGCTTCTGTCACGGCCATGCTGATCGCTGCGCACCCAAGCCTGGGGCCTCTGCAGGCCCCTCCACCGCT • 64 codons fall into groups, the members of each
GTGCAGGTCCACGATGTCTGTGTCTGCCAGCACAACACTGCCGGCCCAAATTGTGAGCGCTGTGCACCCT
TCTACAACAACCGGCCCTGGAGACCGGCGGAGGGCCAGGACGCCCATGAATGCCAAAGGTGCGACTGCAA
group coding for the same amino acid.
TGGGCACTCAGAGACATGTCACTTTGACCCCGCTGTGTTTGCCGCCAGCCAGGGGGCATATGGAGGTGTG
TGTGACAATTGCCGGGACCACACCGAAGGCAAGAACTGTGAGCGGTGTCAGCTGCACTATTTCCGGAACC
• It was originally thought that the genetic code
GGCGCCCGGGAGCTTCCATTCAGGAGACCTGCATCTCCTGCGAGTGTGATCCGGATGGGGCAGTGCCAGG must be the same in all organisms, but
deviations are widespread. E.g. the
mitochondrial genomes and lower eukaryotes
use a nonstandard code.
• Modifications are less common among
prokaryotes. One example is Mycoplasma
species.
The genetic code
The genetic code

• The first two codons are (almost always) specific

for each amino acid.
• The third base is less specific.
• Only tryptophan and methionine have just a
single codon each: all other amino acids are
coded by two, three, four, or six codons. The
genetic code is degenerate.
• Each codon has only one meaning. It codes only
one amino acid. The code is unambiguous.
• The code has a start codon. This codes for
methionine and is AUG.
• The code has three Stop codons: UAA, UAG,
UGA.
• A codon always has an anticodon.
The transfer RNA (tRNA)

(Genomes, T.A. Brown)

The transfer RNA (tRNA)

• Nucleotides: A, C, G, U
• Over 50 modified nucleotides (5-10 in any specific tRNA)
• Nucleotides at some position are completely invariant
(always the same nucleotide) or semi-invariant (always
purine or always a pyrimidine)
• The analysis of the tRNAAla of Saccharomyces cerevisiae
revealed that the molecule adopts a particular base-
paired secondary structure called cloverleaf.
• The 5’ extremity is phosphorylated (almost always pG)
• Acceptor arm: 7 bp. The amino acid is attached to the
adenosine of the CCA terminal sequence.
• The D arm: named after dihydrouridine (always present)
• The V loop: can contain from 3 to 21 nucleotides
depending on the tRNA.
• The TΨC arm named after thymidine-pseudouridine-
cytidine.
• Anticodon arm: three bases (anticodon) that pair with the
(Genomes, T.A. Brown) mRNA
Modified nucleotides

Nucleotides Modified nucleotides

The transfer RNA (tRNA) - structure

(Krahn N., 2020)

The transfer RNA (tRNA) - structure

• Many of the invariant nucleotides positions are

essential in the tertiary structure of tRNA.
• X-ray crystallography studies have shown that
nucleotides in the D (light blue) and TΨC arms
(dark blue) form base pairs that fold the tRNA
into a compact L-shaped structure.
• Each arm of the L-shape is approximately 7 nm
long and 2 nm in diameter.
• The amino acid binding site is at the end of one
arm (green) and the anticodon (pink) at the
end of the other.
• The additional base pairing (dotted lines)
means that the base stacking is almost
continuous from one end of the tRNA to the
other, providing stability to the structure.

(Krahn N., 2020)

The amino acids
Aminoacylation: the attachment of amino acids to tRNAs

Amino acid ATP

pyrophosphate
Adenylated
Amino acid

(Molecular biology of the gene, Watson)

Aminoacylation: the attachment of amino acids to tRNAs

Adenylated Amino acid

Amino acid ATP

pyrophosphate

High energy bond

(Molecular biology of the gene, Watson)

Aminoacylation: the attachment of amino acids to tRNAs

• The reaction is catalysed by a group of

enzymes that is called aminoacyl tRNA
Amino acid ATP
synthases (AARS).
• This is a two-step reaction.
• There are 20 aminoacyl-tRNA synthases.
• There are two classes of aminoacyl-tRNA
Adenylated pyrophosphate synthases.
Amino acid • Class I enzymes attach the amino acid to the
2’–OH group of the terminal nucleotide of
the tRNA
• Class II enzymes attach the amino acid to
the 3’–OH group.
• Aminoacylation is highly accurate.

(Molecular biology of the gene, Watson)

Aminoacyl-tRNA synthetases (AARSs)

• Aminoacyl-tRNA synthetases (AARSs) are the

enzymes that catalyse the aminoacylation
reaction by covalently linking an amino acid to
its cognate tRNA in the first step of protein
translation.
• Step 1: the aminoacyl tRNA synthetase (AARS)
binds to amino acid (AA) and ATP forming ARS-
AA-AMP complex by the release of inorganic
pyrophosphate (PPi)
• Step 2: the ARS-AA-AMP complex then binds the
respective tRNA molecule, thus forming ARS-AA-
tRNA
• Later, the amino acid binds with tRNA at the CCA
end by the release of AMP, and it gets detached
from the AARS.
Aminoacyl-tRNA synthetases (AARSs)

AA • The interaction AARS-aa is less extensive, aa

being much smaller than tRNAs, and presents
Adenylation greater problems concerning specificity
because several pairs of aa are structurally
AA-AMP similar.
Binding of the tRNA to AARS HYDROLISYSIS
• Errors occur at a very low rate for most amino
AA-AMP acids but possibly as frequently as one
tRNA INCORRECT AA AA AMP aminoacylation in 80.
AA-AMP tRNA
• AARS attaches the incorrect amino acid to a
tRNA charging tRNA, this amino acid subsequently being
transformed into the correct one by a second,
AA-tRNA INCORRECT AA HYDROLISYSIS
AA-tRNA separate chemical reaction.
AA AMP • First discovered in the bacterium Bacillus
tRNA
RIGHT aa megaterium for the synthesis of glutamine–
tRNAGln
• AARS has a proofreading activity that involves
AMP AA-tRNA
different contacts with the tRNA.
Aminoacyl-tRNA synthetases (AARSs)
A)
C)
Editing site
Editing site
Acceptor
arm
B) Editing site

Activation site

Activation
Activation
site
site

Anticodon
arm
Aminoacyl-tRNA synthetases (AARSs)
A) C)
Editing site Editing site • Presence of two different active sites in the
Acceptor arm AARSs, one catalytic and another hydrolysing
• “Double sieve” model to explain the
proofreading activity of the AARSs
• The catalytic site of the enzyme acts as the
first sieve that excludes the amino acids that
are large enough or those which do not
Activation site
Activation interact with the active site.
site
• However, smaller amino acids that can fit into
the active site may slip through this first sieve
and may be incorrectly activated. But the
B) Editing site Anticodon editing site is capable of hydrolysing the
arm misactivated amino acids.
• Biochemical and structural studies revealed
that about 50% of the AARSs contain a
Activation site completely separate domain with a hydrolytic
active site for amino acid editing
Codon-anticodon interaction

anticodon

codon

(Genomes, T.A. Brown)

Codon-anticodon interaction: the wobble

Figure © 2010 PJ Russell, iGenetics 3rd ed

Codon-anticodon interaction: the wobble

• Codon–anticodon recognition involves base

pairing between the anticodon of the tRNA and
a codon in the mRNA.
• The first nucleotide of the codon pairs with
nucleotide 36 of the tRNA, the second with
nucleotide 35, and the third with nucleotide 34
• Because the anticodon is in a loop, the triplet of
nucleotides is slightly curved and so cannot
make a uniform alignment with the codon.
• Nonstandard base pair can form between the
third nucleotide of the codon and the first
nucleotide of the anticodon. This is called
“wobble.”
• Wobble reduces the number of tRNAs needed
in a cell by enabling one tRNA to read two or
possibly three codons

Figure © 2010 PJ Russell, iGenetics 3rd ed

Codon-anticodon interaction: the wobble

• In bacteria, G–U base pairs are permitted,

and inosine can pair with A, C, U.
• Instead of needing a different tRNA for each
codon, the four members of a codon family (
e.g. all coding for alanine) can be decoded by
just two tRNAs.
• Wobble reduces the number of tRNAs
needed in a cell by enabling one tRNA to read
two or possibly three codons.
• Bacteria can decode their mRNAs with as few
as 30 tRNAs.
• The human genome has 48 tRNAs. Of these,
16 are predicted to use wobble to decode
two codons each. I-A interaction is weaker.

(Genomes, T.A. Brown)

Codon-anticodon interaction: the wobble

• Prediction: there are 45 tRNAs in human

cells—16 for the wobble pairs and 29
singletons.

• In fact, there are 48 tRNAs. This is because

three codons thought to be decoded as part
of a wobble pair (5’–AAU–3’, 5’–AUC–3’, and
5’–UAU–3’) also have their own individual
tRNAs, although these are present in low
abundance.
Protein posttranslational processing

polypeptide • The genome expression pathway

continues beyond translation.
• The polypeptide that emerges from
the ribosome is inactive.
• Folding
• Proteolytic cleavage
• Post translational modifications

REDO FIGURE

Chemical
folding Proteolytic cleavage
modifications
Protein primary structure

• The primary structure of the

protein is formed by joining amino
acids into a polypeptide.
• Condensation reaction between the
carboxyl group of one amino acid
and the amino group of a second
amino acid.
• One end has a free amino group
and is called the amino, NH2–, or N
terminus;
• The other has a free carboxyl group
and is called the carboxyl, COOH–,
or C terminus.
• The direction of the polypeptide
can therefore be expressed as
either N to C or C to N.
(Genomes, T.A. Brown)
Protein secondary structure

• Secondary structure refers to local folded

structures that form within a polypeptide due
to interactions between atoms of the backbone.
• The two main types of secondary structure are
the a-helix and b-sheet.
• These are stabilized mainly by hydrogen bonds
that form between the carbonyl O of one amino
acid and the amino H of another.
• α helix: the carbonyl (C=O) of one amino acid is
hydrogen-bonded to the amino H (N-H) of an
amino acid that is four down the chain. The R
groups of the amino acids stick outward from
the α helix, where they are free to interact.
• β pleated sheet: two or more segments of a
polypeptide chain line up next to each other,
forming a sheet-like structure held together by
hydrogen bonds. The R groups extend above
(Genomes, T.A. Brown) and below the plane of the sheet.
Protein tertiary structure

• The tertiary structure results from folding the

secondary structural components of the
polypeptide into a three-dimensional
configuration.
• Stabilised hydrogen bonding between
individual amino acids, electrostatic
interactions between the R groups of charged
amino acids, and hydrophobic forces
• Amino acids with non-polar (“water-hating”)
side-groups must be shielded from water by
embedding within the internal regions of the
protein.
• There may also be covalent linkages called
disulfide bridges between cysteine amino
acid residues at various places in the
polypeptide.

(Genomes, T.A. Brown)

Protein quaternary structure

• The quaternary structure involves the

association of two or more polypeptides,
• Each folded into its tertiary structure, into a
multi-subunit protein.
• Not all proteins form quaternary structures.
• Some quaternary structures are held together
by disulfide bridges between the different
polypeptides.
• Proteins can revert to their component
polypeptides, or change their subunit
composition, according to the functional
requirements of the cell
• One example of a protein with quaternary
structure: haemoglobin which carries oxygen
in the blood and is made up of four subunits,
two each of the α and β types.
Proteins have four levels of structure

• Primary structure: formed by

joining amino acids into a
polypeptide.
• Secondary structure: interactions
between atoms of the backbone
• Tertiary structure: folding the
secondary structural components
of the polypeptide into a three-
dimensional configuration.
• Quaternary structure: association
of two or more polypeptides.
Proteins folding

• In the 1960s it was determined that the amino acid

sequence contains all the information needed to
fold the polypeptide into its correct tertiary
structure derives from experiments
• Ribonuclease is a small protein (124 amino acids
long), containing four disulfide bridges and with a
tertiary structure that is made up predominantly of
β-sheet, with very little α-helix.
• Addition of urea, resulted in a decrease in the
activity of the enzyme (measured by testing its
ability to cut RNA) and an increase in the viscosity
of the solution, indicating that the protein was
being denatured by unfolding into an unstructured
polypeptide chain.
• When the urea was removed by dialysis, the
viscosity decreased, and the enzyme activity
reappeared.
• Protein refolds spontaneously when the
(Genomes, T.A. Brown) denaturant (in this case, urea) is removed.
Proteins folding: molecular helpers

There are proteins that help other proteins to fold.

These are called molecular chaperones and have been
studied in most detail in E. coli.
Both eukaryotes and archaea have equivalent proteins,
although some of the details of the way they work are
different.

The molecular chaperones can be divided into two

groups:
• Hsp70 chaperons which include the proteins called
Hsp70 (coded by the dnaK gene in E. coli and sometimes
called DnaK protein), Hsp40 (coded by dnaJ), and GrpE.
• Chaperonins the main version of which is the
GroEL/GroES complex present in bacteria and eukaryotes,
and TRiC found only in eukaryotes.
HSP70 chaperone cycle

(Requena, 2015)
HSP70 chaperone cycle

• The Hsp70 family bind to hydrophobic

regions of unfolded proteins, including
proteins that are still being translated, hold
the protein in an open conformation and
aid folding.
• The Hsp70 protein, as well as binding to the
target polypeptide, has an ATPase activity
and hence can release energy, but it can
function efficiently only
c with the help of
Hsp40 and the Nucleotide Exchange Factor
(NEF) GrpE.
• Hsp40 stimulates the ATPase activity of
Hsp70, and GrpE removes from the
complex the ADP molecule (into which ATP
is converted after energy release) enabling
the cycle to begin again

(Requena, 2015)
Chaperonins GroEL-GroES cycle

Non-native substrate protein

C-terminal tails
Open trans ring - GroEL
GroES
Chaperonins GroEL-GroES cycle
• GroEL and GroES form a multisubunit structure that
looks like a hollowed-out bullet with a central cavity.

• GroEL/GroES complex acts as a cage that prevents the

unfolded protein from aggregating with other
proteins

• This is not the only hypothesis: the cavity unfolds

proteins that have folded incorrectly, passing these
unfolded proteins back toc the cytoplasm so they can
have a second attempt at adopting their correct
tertiary structure.

• Although both the Hsp70 family of chaperones and

the GroEL/GroES chaperonins are present in
eukaryotes, it seems that in these organisms, protein
folding depends mainly on the action of the Hsp70
proteins.
Xu et al., (1997), Nature, 388, 741–750
Protein proteolysis

• Proteolysis is essential for cell survival.

• Usable amino acids derived from protein
degradation are recycled.
• Many newly synthesized proteins, as part of
their maturation, must undergo partial
degradation to achieve their intended biological
functions or be directed to their appropriate
place in the cell.
• Proteins that are damaged
c by reactions with
small highly reactive oxygen–containing
molecules (i.e., reactive oxygen species), or that
undergo other types of chemical modification
must be degraded to prevent their
accumulation, which could prove toxic to the
cell.
• Proteolysis regulates the life span of proteins.

(Donohue, 2003)
Protein proteolysis: the insulin

(Horber,2019)
Protein proteolysis: the insulin

• Several types of virus that infect eukaryotic cells use

them as a way of reducing the sizes of their genomes.
• Polyproteins are not uncommon in eukaryotes.
• Polyproteins are also involved in the synthesis of peptide
hormones in vertebrates.
• Processing occurs with insulin, the protein made in the
islets of Langerhans in the pancreas and responsible for
controlling blood sugar levels.
• Insulin is synthesized as preproinsulin, which is 105
amino acids in length. cc
• The processing pathway involves the removal of the first
24 amino acids to give proinsulin
• This is followed by two additional cuts which excise a
central segment, leaving two active parts of the protein,
• the A and B chains, which link together by formation of
two disulfide bonds to form mature insulin.
Protein Post Translational Modifications (PTMs)

• Many proteins are modified shortly after translation to

mediate proper folding or to direct the nascent protein
to distinct cellular locations (Post Translational
Modifications - PTMs).
• Others occur after folding and localization are completed
to activate or inactivate catalytic activity.
• 5% of the proteome comprises enzymes that perform
more than 200 types of PTMs.
• These enzymes include kinases,
cc phosphatases,
transferases, and ligases, which add or remove functional
groups, proteins, lipids, or sugars to or from amino acid
side chains.
• Many proteins can also modify themselves using
autocatalytic domains, such as autokinase and
autoprotolytic domains.
• PTMs can also be reversible based on the nature of the
modification.
Protein degradation: ubiquitination and the 26S Proteasome

(Stewart H. Lecker, 2006)

Protein degradation: ubiquitination and the 26S Proteasome
• Three enzymes E1, E2, E3 attach ubiquitin molecules,
singly or in chains, to lysine amino acids in proteins
that are targeted for breakdown. There are also
ubiquitin-like proteins, such as SUMO.

• In eukaryotes, the proteasome is a large, multisubunit

structure with a sedimentation coefficient of 26S,
comprising a hollow cylinder of 20S and two “caps” of
19S.

• The entrance into the cavity within the proteasome is

narrow, and a protein must be unfolded before it can
enter.

• The protein is cleaved into short peptides 4–10 amino

acids in length. These are released back into the
cytoplasm, where they are broken down into
individual amino acids, which can be reutilized in
(Genomes, T.A. Brown) protein synthesis.
 SUMMARY

• The genetic code

• The tRNA
• The aminoacylation of the tRNA
• the aminoacyl tRNA synthetase (AARS)
• Codon-anticodon interactions
• Protein structures
• Protein folding
• Protein cleavage
• Post translational modifications
• Protein degradation
Sources:
Genomes. T.A. Brown
Molecular Biology of the Gene. J. Watson
Genetics. P.J. Russel
Aminoacyl-tRNA synthetases. Miguel Angel Rubio Gomez
and Michael Ibba. RNA. 2020