Biochemistry I, Spring Term
Lecture 40
Lecture 40: Protein Synthesis
(mRNA translation) & Protein Export
May 2, 2014
Leader peptide
(export)
HIV Protease
Coding Region
Start codon
Stop codon
Ribosome binding
site
Given the following mRNA molecule, with the
underlined bases representing the ribosome
binding site, identify the correct starting codon.
His-Tag
(purification)
EcoR1
mRNA
Termination
BamH1
Lac Operator
Promoter
i) Which AUG is used to start?
Antibiotic
GAAUUGUGAGCGGAUAACAAUUUCACACAGGAGGAACAGCUAUGAAAGCUUAAUUUAUG...
Resistance
Gene
Origin of
Replication
ii) Given the following tRNAAla molecule. Indicate the anticodon loop
and the location of attachment of the amino acid. What amino acid
would be attached?
iii) If the codon for alanine is 5’GCT3’, what is the anti-codon?
iv) Label the tRNA sites on the ribosome shown on the right.
Protein Synthesis - Overview:
1. The information content of the mRNA is translated
into a polypeptide chain by the ribosome. Three
nucleotide bases, or a codon, encode each amino
acid.
2. mRNA is the template, and synthesis of the
polypeptide chain proceeds in the
aminocarboxy direction as new amino acids
are added to the carboxy terminus of the
growing peptide chain.
H3C
Step I - Chain Initiation:
N-formylmethionine (fMet) and its charged tRNA
function uniquely to initiate chains. The N-formyl
group mimics a peptide bond, allowing the first
methionine to bind to the site on the ribosome that
normally holds the growing peptide.
H
H
S
H
tRNAfMET
O
N
O
N
O
O
N-formyl-Met-tRNAfMET
Normal peptide bond
Steps in formation of Initiation Complex:
i) mRNA first associates with the 30S subunit – the ribosome binding site (also called
the Shine-Dalgarno sequence) forms Watson-Crick hydrogen bonds to a region at the
3' end of 16S rRNA in the 30S subunit of the ribosome.
ii) fMet-tRNAfMet and the 50S subunit combine to form the 70S initiation complex.
30s
fMet
fMet-tRNA-fMet
50s
70s
Initiation
Complex
UAC
fMet
RBS-AUG-----AAA-----GCU-----UAA
mRNA
1
RBS-AUG-----AAA-----GCU-----UA
A
UAC
RBS-AUG-----AAA-----GCU-----UAA
fMet
UAC
RBS-AUG-----AAA-----GCU-----UAA
Biochemistry I, Spring Term
Lecture 40
May 2, 2014
Step II - Chain Elongation: (Synthesis of Met-Lys-Ala; mRNA = SD-AUG-AAA-GCUUAA) RBS = ribosome binding site)
a) fMet-tRNA occupies the P site in the
first cycle. Growing protein is found here
at the beginning of subsequent cycles.
b) aminoacyl-tRNA-Lys (next AA to be
added) is brought to the A site.
c) Peptidyl transfer, or transpeptidation
occurs - the free amino group of the
amino acid in the A-site performs a
nucleophilic attack on the carbonyl group
of the amino acid (or peptide) attached to
the tRNA in the P-site. No energy is
required to form the peptide bond.
d) Translocation of the ribosome occurs.
e) The uncharged tRNA leaves the E site.
a
fMet
Lys
UAC
RBS-AUG-----AAA-----GCU-----UAA
b
UUU
fMet
Lys
Before Peptide Bond Formation
UAC
UUU
RBS-AUG-----AAA-----GCU-----UAA
tRNA-C-CO
c
H
Lys-fMet
d
tRNA-C-CAdenine
O
O OH
N
fMet
H
H
UUU
RBS-AUG-----AAA-----GCU-----UAA
Lys-fMet
O
O OH
N
Lys
A-Site
After Peptide Bond Formation
tRNA-C-C-
e
O
H
P-Site
UAC
Adenine
O
O
UAC
UUU
RBS-AUG-----AAA-----GCU-----UAA
Adenine
tRNA-C-C-
OHOH
UAC
H
Ala
O
O OH
N
Lys
fMet
O
O
Lys-fMet
P-Site
CGA
Adenine
O
N
H
H
A-Site
UUU
RBS-AUG-----AAA-----GCU-----UAA
tRNA-CC
O
Step III - Chain Termination:
Protein release factor (which is a protein
that looks like a tRNA) and a stop codon are
required to finish the polypeptide chain.
Water hydrolyzes the peptide from the Psite, releasing the complete tri-peptide. The
mRNA is released and the ribosome 30s
and 50s subunits dissociate.
2
O OH
O
tRNA-C-C-
Adenine
O
Adenine
OHOH
ALA
O
N
H
Lys
CGA
H
N
O
fMet
N
H
O
O
O
H
CGA
ALAO
N
H
H
N
Lys
O
fMet
N
H
P-Site
Ala-Lys-fMet
CGA
SD-AUG-----AAA-----GCU-----UAA
CGA
SD-AUG-----AAA-----GCU-----UAA
O
H
Biochemistry I, Spring Term
Lecture 40
May 2, 2014
B. Export of Recombinant Proteins.
A specialized protein sequence, called the
Leader peptide
(export)
leader peptide, when present as the amino
HIV Protease
His-Tag
terminal residues of the protein, signals the
Start codon
Coding Region
(purification)
export of the protein out of the cell. This may
Stop codon
Ribosome binding
EcoR1
site
mRNA
reduce the toxicity of the protein and make
Termination
Lac Operator
BamH1
purification easier since only a small number
of native proteins are exported out of the cell.
Promoter
During the export process, this peptide is cleaved by the
leader peptidase (also known as signal
peptidase), producing the mature exported peptide.
Antibiotic
The main features of this peptide are:
Resistance
Origin of
Gene
i) Basic residue at amino terminus
Replication
ii) Non-polar segment of ~15 amino acids.
iii) Cleavage site, which is followed by another basic residue (---Ala^Arg---)
A) The complete DNA sequence of expression plasmid, from the promoter to the stop codon is:
-35
-10
mRNA Start
RBS
Start-----Leader peptide-----TTGACATTTATGCTTCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAGGAACAGCTATGAAACAATCCACAATTGCGCTCGCGCTT
Promoter
Lac operator
fMetLysGlnSerThrIleAlaLeuAlaLeu
-----Leader peptide (cont)----------------|---HIV---Protease-|-----|--His Tag-------|
CTCCCGCTACTATTTACTCCAGTGACCAAAGCGCGCGAATTCCCTCAGATC-TTAAATTTCGGATCCCACCACCACCACCACCACTAA.....
LeuProLeuLeuPheThrProValThrLysAlaArgGluPheProGlnIle-LeuAsnPheGlySerHisHisHisHisHisHisStop
^
EcoR1
BamHI
B) The peptide, as it comes off of the ribosome will look like:
-------------------------Leader Sequence---------------------------------|----HIV protease--| |His Tag..
fMetLysGlnSerThrIleAlaLeuAlaLeuLeuProLeuLeuPheThrProValThrLysAlaArgGluPheProGlnIle..LeuAsnPheGlySerHis6
^
C) After export out of the cell the final product will be as follows (The bold amino acids were added as
part of the EcoR1 and BamH1 sites that were required to insert the PCR product into the expression
vector.):
|----HIV protease----|
|----His Tag-------|
ArgGluPheProGlnIle----LeuAsnPheGlySerHisHisHisHisHisHis
Protein Export Machinery
1. mRNA binds to the ribosome.
2. When leader peptide emerges from the ribosome it binds to the signal recognition particle (SRP).
3. The ribosome/mRNA/SRP binds to transport machinery(translocon) in the cell membrane.
4. Protein synthesis continues, protein extruded outside the cell.
5. Leader peptidase cleaves off leader peptide.
6. Protein is completely exported out of the cell.
2
1
Inside
bacterial
cell
mRNA
ribosome
SRP
NH2
Cell
Membrane
50s
3
6
5
4
Outside
30s
mRNA
3
translocon
NH2
leader peptide
NH2
Arg
Ala
NH2
Leader
peptidase
cleavage
Ala
Arg
NH2
NH2
HisHisHisHisHisHis
Biochemistry I, Spring Term
Lecture 40
May 2, 2014
C. Affinity Purification:His6-tag binds to immobilized nickel ions on
Culture media
(exported HIV
protease +
contaminants)
chromatograph beads – allowing one-step purification of HIV protease from
culture media via affinity chromatography. Elution with
imidazole (competes
with His sidechain for
N
N
Ni+).
N
H
imidazole
Cell ← Wash → Elute
lysate
O
HN
N
H
histidine
column
beads Ni+
Ni+
N
Ni+
N
O
NH
N
Ni+
O
N
NH
His-tag
(HIV Protease)
Summary of Expression Features:
Promoter
-35 -10
DNA
Lac
Operator
Ribosome
binding site
Start
codon
Leader
peptide
HIV Protease
Coding Region
His-Tag
Stop
codon
mRNA
Termination
mRNA
Protein
Protein
(exported)
Promoter
mRNA
production
Protein
Production
Protein
Export
Protein
Purification
X
Lac O
RBS
Start
codon
Leader
codons
HIV P
codons
His-tag
codons
Stop
codon
mRNA
term
X
X
X
X
X
X
X
X
X
X
Comparison of Expression Hosts:
Host
Advantages
Disadvantages
Typical Use
Bacteria (E.
Coli)
 Easy DNA
manipulation
 High protein yields
 Similar posttranslational
modification as
mammals.
 No toxic impurities.
 Equivalent protein
modification as
human proteins.
 No toxic impurities
 Lack of post-translational
modification (e.g. glycosylation)
 Toxic impurities.
 DNA manipulations more
difficult.
 Lower protein yield (typically,
but not always).
 Laboratory research
 Diagnostic reagents
 DNA manipulations more
difficult.
 Cell culture expensive.
 Protein yields can be low.
 Proteins that are used as
drugs (e.g. human growth
hormone, antibodies.)
Yeast
Mammalian
cells
T7 Expression system – also widely used.



4
T7 Promoter 5’ to coding region is only
recognized by T7 RNAP.
Gene for T7 RNAP is located on
chromosome, under control of lac.
Addition of IPTG induces production of T7
RNAP, followed by production of protein
from T7 promoter – only protein that is
made is the desired protein.
 Laboratory research
 Food processing enzymes