Protein translation and post

translational modification
 Learning Objectives

At the end of this lecture the students will be able to:

• Outline the mechanisms by which ribosomes can translate a
mRNA sequence into a protein sequence
• Describe the role of aminoacyl tRNAs in ensuring the fidelity of
the genetic code
• State how a ribosome recognises the start and end of a sequence
to be translated
• Explain why some antibiotics inhibit protein synthesis in
prokaryotes but not eukaryotes
• Identify the features of a newly-synthesised protein that are
required for it to enter the secretory pathway
• Give examples of the ways in which newly-synthesised proteins
can be post-translationally modified e.g. insulin
The central dogma

DNA

RNA

Protein
 Replication

DNA
Transcription
RNA
Translation

Protein
• There is a linear relationship between the information
encoded within DNA and the proteins which are
synthesised using that information
• Eukaryotic DNA contains introns (non-coding) and
exons (coding)
• Transcription of DNA -> pre-mRNA -> SPLICING
(removal of introns) -> mRNA

Exon DNA 300nt

via mRNA
Protein 100 amino acids
Three nucleotides encode one amino acid
A group of three nucleotides is called a codon
 Structure of a typical mRNA

7MeG AAAAAn
5’cap 5’UTR coding region 3’UTR polyA

5’ “cap” (7-Methyl Guanosine) – entry site for ribosome

UTR – untranslated region
polyA – protects mRNA
 The genetic code (U=RNA, T=DNA)
second base
U C A G
UUU UCU UAU UGU U
UUC UCC UAC UGC C
U UUA UCA UAA UGA A
UUG UCG UAG UGG G
CUU CCU CAU CGU U
CGC C
first base C CUC
CUA
C CC
C CA
CAC
CAA CGA A “wobble”
CUG C CG CAG CGG G position
AUU A CU AAU A GU U
A GC C
A AUC
AUA
A CC
A CA
AAC
AAA A GA A
AUG A CG AAG A GG G
GUU GCU GAU GGU U
GG C C
G GUC
GUA
GCC
GCA
GAC
GAA GG A A
GUG GCG GAG GG G G
 There are 64 codons for 20 amino acids
second base
U C A G
Phe Ser Tyr Cys U
Phe Ser Tyr Cys C
U Leu Ser STOP STOP A
Leu Ser STOP Trp G
Leu Pro His Arg U
Leu Pro His Arg C
C Leu Pro Gln Arg A
“wobble”
first base Leu Pro Gln Arg G
Ile Thr Asn Ser U position
Ile Thr Asn Ser C
A Ile Thr Lys Arg A
Met Thr Lys Arg G
Val Ala Asp Gly U
Val Ala Asp Gly C
G Val Ala Glu Gly A
Val Ala Glu Gly G
 U C A G
Phe Ser Tyr Cys U Stop =
Phe Ser Tyr Cys C UAA
U Leu Ser STOP STOP A
UAG
Leu Ser STOP Trp G
Leu Pro His Arg U UGA
Leu Pro His Arg C
C Leu Pro Gln Arg A
Leu Pro Gln Arg G Rare
Ile Thr Asn Ser U
Ile Thr Asn Ser
amino
C
Start
A Ile Thr Lys Arg A acids have
Met Thr Lys Arg G fewer
(Met) = Val Ala Asp Gly U codons
AUG Val Ala Asp Gly C
G Val Ala Glu Gly A
e.g. Met,
Val Ala Glu Gly G Trp
 Reading a mRNA

[5’cap]CCGGACUACCUCUGGACCCCCTCCCCU
GUCCCCAACGCUGAGCCGAAACC AUG CAU
GGG CGC CUG AAG GUA AAG ACG UCG GCU
GAA GAG CAG GCA GAG GCC AAA AGG CUA
GAA CGA GAG AAG CUA AAG CUC UAC CAG
UGA GCCACUCAAGCUGUCUUCAAAAn 3’

• Ribosome scans from 5’ end of mRNA (cap)

• Translation starts at first AUG, continues in frame, i.e. with
immediately succeeding triplet codon (CAU) and so on…..
• Translation stops at first in frame termination codon
Messenger RNA (mRNA) is transcribed
from DNA (pre-mRNA) and processed
(spliced) in the nucleus
mRNA is transported out of the nucleus
and translated in the 5’->3’ direction into
protein (N->C direction) in the cytoplasm
and on the rough endoplasmic reticulum
The machinery for translating mRNA is
the ribosome, a large 2 subunit complex
of proteins and ribosomal RNA (rRNA)
 S = Svedberg unit of
sedimentation in a centrifuge
(size)
Nobel Prize Chemistry 2009;
Yonath, Steitz and
Ramakrishnan
transfer RNA: (>1 3´ end Amino acid (Met)
for each aa) the 5´ end
transporter of
amino acids to the
ribosome

antiparallel
binding - like Anticodon
DNA UAC
AUG Codon (wobble)
5’ 3’ mRNA
 Aminoacyl tRNA synthetases
• one for each aa
• important role in fidelity of translation
Amino Acid (selectivity for correct aa; hydrolysis of
incorrect aa-tRNA)
ATP
E
Amino Acid
PPi

E -AMP-Amino Acid

(Adenylated aa) AMP E

Mutated in cancers, neuropathies, autoimmunity, metabolic disease

 Translation: a) Initiation

Step 1: dissociation of ribosome subunits (40S + 60S)

Step 2: assembly of preinitiation complex

containing Met-tRNA + eIFs + 40S subunit

Step 3: binding of mRNA to preinitiation complex

Step 4: binding of 60S subunit

(simplified version; for details see Alberts et al., Molecular Biology

of the Cell, 4th ed.)
 Translation: a) Initiation
Step 2: assembly of pre-initiation complex
Initiation
Met
Factors:
eIF-2 Small Ribosomal
40S
GTP UA C Subunit

eIF4E, G
[5’cap] U G C G G A U G C G A U G G AAA U U C 3’ mRNA

• Only initiator Met-tRNA can bind to 40S subunit alone

• 40S subunit is primarily involved in tRNA and mRNA recognition
 Translation: a) Initiation

Step 3: binding of mRNA to preinitiation complex;

initiator Met binding sets the frame of the translation

Met

40S
eIF4E, G UA C
[5’cap] U G C G G A U G C G A U G G AAA U U C 3’ mRNA
eIF-2 GTP

• eIF4E and G bind to cap and are recognised by 40S/Met-

tRNA/eIF2
 Translation: a) Initiation
Step 4: binding of 60S subunit

GTP GDP + Pi (ensures correct base pairing)

Met

40S
UA C
UGCGG A U G C G A U G G AAA U U C 3’ mRNA
eIF-2 GTP GDP
60S
 Translation: a) Initiation

Step 4: binding of 60S subunit

eIF-2 GDP
GTP GDP + Pi

Met

40S
UA C
UGCGG A U G C G A U G G AAA U U C 3’ mRNA

60S
 Translation: b) Elongation

Step 1: binding of new tRNA carrying second

amino acid to A (amino acyl) site

Step 2: catalysis of peptide bond between

the two amino acids by peptidyl transferase

Step 3: translocation of tRNA to P (peptidyl) site

and dissociation of first tRNA
 Translation: b) Elongation

Step 1: binding of new tRNA to immediately adjacent A

site in frame with initiator Met

Met Arg

UA C G C U
UGCGG A U G C G A U G G AAA U U C 3’ mRNA

P site A site
 Translation: b) Elongation
Step 2: catalysis of peptide bond between the two
amino acids by peptidyl transferase on 60S subunit

PT
Met Arg

UA C G C U
UGCGG A U G C G A U G G AAA U U C 3’ mRNA

P site A site
 Translation: b) Elongation

Step 2 continued:

Met
Arg

UAC GCU
UGCGG A U G C G A U G G AAA U U C 3’ mRNA

P site A site
 Translation: b) Elongation

Step 3: translocation of peptidyl tRNA to P site

Met
Arg

UAC GCU
UGCGG A U G C G A U G G AAA U U C 3’ mRNA

P site A site

• Elongation Factors (EFs) are proteins that promote

movement of ribosome along mRNA using GTP
 Translation: b) Elongation

Step 3 (continued): dissociation of first tRNA

Met
Arg

UAC
GCU
UGCGG A U G C G A U G G AAA U U C 3’ mRNA

P site A site
 Translation: b) Elongation

New cycle with new step 1

Met
Arg Trp

G C U AC C
UGCGG A U G C G A U G G AAA U U C 3’ mRNA

P site A site
 Translation: b) Elongation

New step 2

Met
Arg
Trp

G C U AC C
UGCGG A U G C G A U G G AAA U U C 3’ mRNA

P site A site
 Translation: b) Elongation

New step 3

Met
Arg
Trp

G C U AC C
UGCGG A U G C G A U G G AAA U U C 3’ mRNA
P site A site
 Translation: b) Elongation

Etc., etc.

Met
Arg
Trp

GCU
AC C
UGCGG A U G C G A U G G AAA U U C 3’ mRNA
P site A site

EFs use the energy of GTP to enhance the efficiency and accuracy of
translation by providing “pauses” (e.g. GTP hydrolysis) that allow
incorrect base pairs to dissociate
 Translation: c) Termination

Step 1: recognition of stop codon

Step 2: release of peptide chain

Step 3: dissociation of release factors and ribosomes

 Translation: c) Termination

Step 1: recognition of stop codon

Gln
Release factors
(proteins, not tRNAs)
RF bind to empty A site
GU C
CG U G A A U C A G U AA U U C G AA U 3’ mRNA
Out of frame UGA
 Translation: c) Termination

Step 2: release of peptide chain

Gln

RF

GUC
CG U G A A U C A G U AA U U C G AA U 3’ mRNA

Peptidyl transferase catalyses transfer of the completed

protein chain to water and releases it from the ribosome
 Translation: c) Termination

RF

Gln GUC 40S

CG U G A AUC A G U AA U U C G AA U 3’ mRNA

60S
Protein Translation animation

http://www.youtube.com/watch?v=5bLEDd-PSTQ
 Polyribosomes

Ribosomes do not work singly on a mRNA but in

multiple copies on the mRNA – a polyribosome – like
a string of beads

http://www.sumanasinc.com/webcontent/animations/content/
polyribosomes.html
 Translation speed

• Translation speed of each ribosome = 15 amino

acids/sec

• Multiple ribosomes processing simultaneously a

300 a.a. long protein, i.e. one ribosome every 30a.a.
of synthesized protein - the number of protein
molecules produced in 1 min is ~4000
 Many antibiotics inhibit protein
synthesis in prokaryotes
• Translational machinery is complex, easily disrupted –
common target for antibiotics

• Antibiotics exploit differences between prokaryotic and

eukaryotic ribosomes and translation factors
• Antibiotics selectively inhibit prokaryotes

• Antibiotics are natural products of bacteria or fungi to

give them a selective advantage over other microbes
 Antibiotics inhibiting protein synthesis

• Streptomycin Inhibits initiation

• Tetracycline Inhibits aa-tRNA binding
• Erythromycin Inhibits translocation
• Chloramphenicol Inhibits peptidyl
transferase
• Puromycin Terminates elongation
prematurely
INTRACELLULAR Plasma membrane
Lysosome
COMPARTMENTS
Golgi Apparatus
Protein synthesis takes
place in the cytoplasm Rough Endoplasmic Reticulum
Ribosome
Vacuole
Nucleus
Most cellular Peroxisome
compartments are Nucleolus
bounded by a Mitochondrion
membrane so the cell Smooth Endoplasmic Reticulum
needs a mechanism to Cytosol
transfer proteins across
membranes
 Secretory and transmembrane proteins are
synthesised in the Rough Endoplasmic Reticulum

Same
ribosomes on
RER as for
cytoplasmic
proteins
 Protein synthesis on
Rough Endoplasmic Reticulum (RER):
secreted and transmembrane proteins
First 20-24 amino acids = “signal sequence”
(hydrophobic amino acids, e.g. Leu, Ile,
Phe, Trp, Tyr, Ala)

40S
mRNA
60S
 Protein synthesis on RER

Step 1: recognition of signal cytoplasm

sequence by a protein-RNA complex
“Signal-Recognition Particle” (SRP),
halting translation
RER
SRP

lumen
40S
mRNA
60S SRP
Receptor
 Protein synthesis on RER

Step 2: binding of SRP to a receptor at

the RER surface, translation resumes
RER

SRP

40S

60S SRP
Receptor
 Protein synthesis on RER

Step 3: translocation
into the lumen of RER
RER

SRP

40S

60S
Protein channel
 Protein synthesis on RER

Cytoplasm
RER lumen
40S

60S
Secreted protein

Transmembrane
proteins have an extra
hydrophobic
sequence holding
them in the
membrane
Protein synthesis on RER

Step 4: cleavage of
signal sequence by
signal peptidase (co-
translational) and
folding
 Post-translational modification
- After synthesis most proteins are modified further before
they are fully functional
- Only 20 amino acids – cell uses post-translational
modifications (over 200) to increase diversity, including:
• Disulphide bond formation (e.g. insulin)
• Proteolytic cleavage (e.g. insulin -> A and B chains)
• Addition of carbohydrate (Glycosylation)
• Addition of phosphate (Phosphorylation)
• Addition of lipid groups (Prenylation, Acylation)
• Hydroxylation (e.g. Collagen; Leitinger lecture)
Insulin biosynthesis
in pancreatic β cells
Insulin undergoes extensive post-
translational modification along
the production pathway, including
disulphide bond formation in the
ER and proteolytic cleavage in the
secretory vesicle to produce
active insulin
 Glycosylation in the RER and Golgi complex

Pre-assembled carbohydrate Carbohydrate chains

chains N-linked to Asn of processed by trimming
(AsnXSer/Thr) “sequon” followed by extension

RER Golgi

PM

Transport
Glycan on
lipid-linked vesicles
precursor

Initial N-glycosylation Glycan processing ( )