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MOLECULAR BIOLOGY – Protein synthesis
PROTEIN SYNTHESIS
TRANSLATION
MOLECULAR BIOLOGY – Protein synthesis
The Genetic Code
The nucleotide sequence of mRNA contains three letter codons that specify all of
the 20 amino acids found in proteins plus a signal to terminate protein synthesis
The order that the codons appear in the mRNA (5’ - 3’) directly dictates the order of
the amino acids in the polypeptide chain of the protein (N - C termini)
Figure 6-50 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
DIFFERENT READING FRAMES OF
mRNA THE SAME SEQUENCE
Genetic code can be read in 3 ways depending upon
where you start!
+1 frameshift
+2 frameshift
The genetic information encoded in each reading frame is different
Figure 6-51 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
How is the mRNA genetic code read during protein synthesis?
Transfer RNAs (tRNA) act as adapters between the mRNA and protein
synthesising machinery (‘ribosomes’)
As each specific tRNA (i.e. defined by its anticodon) is bound to a specific amino acid at its 3’ end,
according to the genetic code in the mRNA, is recruited to the ribosome
tRNA triplet nucleotide sequences that are complementary to mRNA codons, called ‘anticodons’,
form specific base-pairs with the mRNA codons
Figure 6-52 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
The minimum set of required tRNAs is 31 but there are 61 possible
amino acid coding codons !
Some tRNA can read more than one codon !
This is because the first base of the anti-codon (that binds to the third
base of the mRNA codon) is not squeezed/ constrained as it would be
in a DNA double helix and can wobble making other base pairings
possible i.e. ‘wobble base-paring‘
Therefore a single tRNA can two
recognise two different codons for
the same amino acid !
Adenosine to inosine conversion at the
wobble position of the anticodon in some
tRNAs permit it to recognise three different
codons !
Figure 6-53 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
tRNA structure summary video/ tutorial
http://www.youtube.com/watch?v=4MRCH_J7Fhk
MOLECULAR BIOLOGY – Protein synthesis
Attachment of amino-acids to tRNAs (‘Charging’)
Each tRNA is charged by a specific enzymes that recognise both the tRNA
and the amino acid - called ‘aminoacyl tRNA synthetases‘
e.g. tryptophanyl tRNA synthetase
Charging is a two step process
Figure 6-58 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
tRNA charging
Uncharged tRNA
2. Transfer of the amino
acid to the free 3’OH of
the tRNA
(Aminoacyl-AMP)
1. Amino acid
adenylation
Charged tRNA
Figure 6-56 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
tRNA amino acid charging video/ tutorial
http://www.phschool.com/science/biology_place/biocoach/translation/addaa.html
MOLECULAR BIOLOGY – Protein synthesis
During protein synthesis tRNAs are sequentially released from
their corresponding amino acids
peptide bond
What is responsible for the formation of
peptide bonds within the cell ?
Figure 6-61 Molecular Biology of the Cell (© Garland Science 2008)
amino (N-) terminus
carboxyl (C-) terminus
MOLECULAR BIOLOGY – Protein synthesis
Very large protein-RNA complexes called ‘Ribosomes’
Ribosome comprise one large
and one small subunit
Ribosomes bind both the mRNA and amino acid charged tRNAs to decode the information in the mRNA
into a polypeptide sequence of amino acids
Figure 6-63 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
Ribosomal RNA (‘rRNA’) critical to ribosome function
rRNAs:
• 2/3 of the molecular weight for ribosome
(prokaryotes)
• form complex and defined secondary structure
• originally thought to have structural role, now
known to required for most of the ribosome’s
functions
• X-ray crystallography show no proteins are
proximal to catalytic site to participate in peptide
bond formation
• 23S rRNA (prokaryotes) acts as a ‘peptidyl
transferase’ ribozyme
• sequence mutagenesis studies of 23S rRNA
Prokaryotic 16S rRNA
show its function is to correctly position the
incoming charged tRNA to allow spontaneous
formation of the peptide bond
MOLECULAR BIOLOGY – Protein synthesis
3D ribosomal structure (70S prokaryotic)
The interface between
large & small s/u’s form a
groove for mRNA binding
and three tRNA binding
sites: A (acceptor), P
(peptide) & E (exit)
Figure 6-64 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
Correctly identifying the translation start-point in mRNA
Prokaryotic ribosomes
Translation always starts at an AUG codon (coding for methionine) called the ‘start codon’
How does the
ribosome
recognise the
correct AUG as
the start codon ?
Various ‘initiation
factors (IFs)’
participate in this
process
Shine-Delgarno
sequence
N-Formyl methionine charged tRNA is then recruited
into the P-site ready for translation to start
7bp
nnnnnnAGGAGGUnnnnnnnAUGnnnnnnn
start codon
UCCUCCA
Enables translation of polycistronic mRNAs
Shine-Delgarno sequence
mRNA
16S rRNA base-pairing leads to small
ribosomal s/u recognition, large s/u
recruitment and formation of the ‘70S
initiation complex’
Variations in the S-D sequence can effect translation
initiation efficiency
MOLECULAR BIOLOGY – Protein synthesis
Prokaryotic translation summary video/
tutorial (including inititation)
http://www.biostudio.com/d_%20Protein%20Synthesis%20Prokaryotic.htm
MOLECULAR BIOLOGY – Protein synthesis
Eukaryotic ribosomes
The 5’ cap structure of the mRNA is recognised leading to the recruitment of the 40S small
ribosome s/u and the initiator tRNAmet and this initiator complex ‘scans’ in a 5’ to 3’ direction until the
first AUG is recognised
eIF3 (small 40S ribosome
s/u binding)
m7G 5’-cap
eIF2 (initiator tRNAmet binding)
Small 40S
ribosomal s/u
eIF4 (cap binding)
‘ribosome scanning’
‘Eukaryotic initiation
factors (eIFs)’ facilitate
the process
N.B. the sequence context of
the AUG is important
(consensus
GCCRCCAUGG) meaning
some AUG‘s maybe skipped
Figure 6-72 (part 2 of 5) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
Ribosome scanning leads to the selection of the appropriate start codon/ AUG
eIF5 assisted
Ribosome now correctly placed to ‘read the correct frame in the mRNA
Figure 6-72 (part 3 of 5) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
Elongation phase of translation
Bound tRNAs move to
next site (A-P or P-E)
Charged tRNA
enters A-site.
Specificity
dictated by
codon-anticodon
base-pairing
New peptide bond
formation
(between adjacent
amino acids in P
& A-sites)
As next charged tRNA enters Asite the E-site occupant departs
the ribosome
Ribosome
‘translocates’
along mRNA to
next codon
N.B. The A- and E-sites can never be simultaneously occupied
Figure 6-66 Molecular Biology of the Cell (© Garland Science 2008)
The elongation phase of translation
is essentially similar in prokaryotes
and eukaryotes involving a repetition
of a series of steps
MOLECULAR BIOLOGY – Protein synthesis
The elongation phase is governed ‘elongation factors (EFs)’
Prokaryotic example used below (eukaryotes have other EFs but principle is the same)
‘EF-Ts’ exchanges GDP
from EF-Tu for fresh GTP
allowing it to recruit more
charged tRNAs to the Asite
EF-Ts
GDP
‘EF-Tu’ binds to charged tRNAs
and delivers them to the A-site.
This requires energy from GTP
hydrolysis to GDP
Figure 6-67 (part 1 of 7) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
Ribosomal TRANSLOCATION
along the mRNA and the
associated migration of the t-RNAs
from the A- to P-site or P- to E-site
also requires energy from GTP
hydrolysis mediated by ‘EF-G’
EF-G binding causes bound tRNAs
to exist partially bound to both
sites (A & P or P & E) and GTP
hydrolysis completes the
translocation
Figure 6-67 (part 6 of 7) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
Termination of translation
1. No tRNAs can recognise a stop codon in the A-site. The stop codon is therefore
recognised by a ‘release factor (RF)’ (either RF1 or RF2 depending on stop codon
sequence)
2. RFs activate the peptidyl-transferase of the ribosome to hydrolyse the bond between
the completed polypeptide chain and the tRNA in the P-site
3. Further RFs (RF3 and ‘Ribosome recycling factor (RRF)’ dissociate RF1/2 and the
small/ large ribosomal s/u’s
MOLECULAR BIOLOGY – Protein synthesis
>1 ribosome can translate a single mRNA at a time
‘Polysome (i.e. polyribosome)’ formation in eukaryotes
The 5’ cap binding protein (eIF4)
interacts with PABP (poly A-binding
protein) at the 3’ end of the mRNA with
translating ribosomes at approx 100bp
intervals around the length of the mRNA
transcript
Transmission electron micrograph
N.B. In eukaryotes
the mRNA is
extensively
processed in the
nucleus before
being exported into
the cytoplasm for
translation
Figure 6-76 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
In bacteria mRNA transcription and translation are coupled in the
cytoplasm !
Even as the mRNA sequence is
appearing from the RNA polymerase
complex it is recognised by ribosomes
and immediately translated into protein !
N.B. the mRNA transcripts are
often polycistronic coding for more
than one protein !
MOLECULAR BIOLOGY – Protein synthesis
Although mechanistically similar, prokaryotic and eukaryotic
ribosomes are not identical
MANY ANTIBIOTICS WORK BY INHIBITING BACTERIAL PROTEIN SYNTHESIS
30S
Figure 6-79 Molecular Biology of the Cell (© Garland Science 2008)
50S
MOLECULAR BIOLOGY – Protein synthesis
Small molecule inhibitors of protein synthesis
Table 6-4 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein synthesis
Translation summary video/ tutorial
http://bcs.whfreeman.com/thelifewire/content/chp12/1202003.html
MOLECULAR BIOLOGY – Protein structure & function
PROTEIN STRUCTURE AND FUNCTIONS
MOLECULAR BIOLOGY – Protein structure & function
Proteins are polymers of different amino acids joined by peptide
bonds
Amino acids
Protein
synthesis
Polypeptide (i.e. protein)
Each amino acid has a different chemical side chain and the order of these side chains in a
protein sequence is what conveys its structure and functionality
Figure 3-1 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
Grouping the 20 amino acids by their chemical properties
Hydrophylic
Hydrophobic
Learn the amino acid abbreviations and properties
Figure 3-2 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
Types of chemical bonding contributing to protein structure
formation
Non covalent bond/ interactions
covalent peptide bond
N.B. Non covalent bonding can exist between
any combination of the amino acid side chains
and the peptide backbone
Figure 3-4 Molecular Biology of the Cell (© Garland Science 2008)
covalent disulphide bond
(between two Cys side chains)
MOLECULAR BIOLOGY – Protein structure & function
Primary (1o)
The simple order of the
amino acids in the
polypeptide chain
Secondary (2o)
Interactions between the amino acid
(mostly hydrogen bonds) resulting in an
array of regular sub-structures ( helix
strand)
Tertiary (3o)
Quaternary (4o)
The relative arrangement
of multiple proteins (i.e.
tertiary structures) in a
complex i.e. sub units
The overall 3D
structure of a protein
describing the spatial
arrangement of the
secondary structural
elements (themselves
often found in discreet
motifs)
Amino acid side chain interactions Covalent peptide
bonding
Chemical bonding results in 4 levels of protein structure
MOLECULAR BIOLOGY – Protein structure & function
Type of 2o protein structure
Alpha-helix
A right handed coiled conformation in
which the NH group of an amino acid in
the peptide backbone forms a hydrogen
bond (shown in yellow and pink
opposite)with the CO group of an
amino acid 4 residues earlier
Beta-strand (leading to beta sheets)
The polypeptide chain exists in a stretched
conformation and peptide backbone hydrogen bonds
form between the NH and CO groups (light blue) of
amino acids in different strands
As the polypeptide chain has polarity (i.e. an N- and Cterminus) the two strands can run PARALLEL or ANTIPARALLEL to each other
The arrangement of beta strands forms a beta sheet
MOLECULAR BIOLOGY – Protein structure & function
2o structural elements within a solved protein 3o structure (e.g.
using X-ray crystallography) are often represented by ‘ribbon
diagrams’
Alphahelix
Anti-parallel
beta-sheet
Parallel beta-sheet
e.g. dihydrofolate reductase
MOLECULAR BIOLOGY – Protein structure & function
Newly translated proteins must ‘fold’ to attain functional structure
Chemical properties of the amino acid side chains and primary protein structure
contribute to the spontaneous folding pattern
Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
Protein folding and forming a functional structure is complex !
Correct incorporation of essential ‘cofactors’
during polypeptide chain folding e.g. metal ions in
enzymes, rRNAs in ribosomes
Addition of ‘post-translational’ covalent
modifications required for protein activity or
recruitment of other proteins e.g. phosphorylation
Successful assembly of multi-protein complexes
required to attain functionality e.g. ribosome
assembly
Figure 6-82 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
The cell’s cytoplasm is ‘molecularly crowded’
CYTOPLASM IS TIGHTLY PACKED
WITH MOLECULES
Cytoplasm
PROBLEM: How do newly produced polypeptide
chains fold appropriately without forming aggregates
with the ‘molecular crowd’ of other proteins in the
cytoplasm?
MOLECULAR BIOLOGY – Protein structure & function
Chaperonins/ chaperons needed for proper folding
Chaperonins/ Chaperons:
• proteins within the cell that assist with appropriate folding of proteins
• their role is to prevent misfolding rather than actively direct correct folding
• can act to delay any folding (e.g. as the nascent polypeptide chain emerges
from the ribosome)
• can also ‘rescue’ misfolded proteins to the correct folding conformation
Chaperonins/ chaperons exist to
ensure that nothing inappropriate
occurs ! . . . (in a protein folding
sense).
MOLECULAR BIOLOGY – Protein structure & function
e.g. Heat Shock Protein 70 (Hsp70)
1. Hsp70-ATP able to loosely bind
hydrophobic patches of amino
acids as they emerge from the
ribosome
2. Peptide binding induces
intrinsic ATPase activity in
HSP70
3. Hsp70-ADP tightly associates
with unfolded protein and
protects it from aggregating
5. Protein spontaneously folds
into correct confirmation
4. Nucleotide exchange factors
eventually replace the ADP with
ATP and HSP70 releases the
unfolded protein
6. A small percentage of protein
incorrectly folds
The expression of Hsps (heat shock proteins) increases as temperature increases because folded
proteins are more likely to unfold/ denature at higher temperatures
Figure 6-86 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
Multi-subunit complex
‘cocktail shaker’
e.g. GroEL/ Hsp60 ‘rescues’ misfolded proteins
1. Misfolded proteins with exposed
hydrophobic regions bind hydrophobic
regions in the neck of the GroEL
3. Hydrolysis of the bound ATP (plus binding of
additional ATP) releases the GroES cap and the
correctly folded protein
2. The binding of the GroES cap and
ATP cause conformational change that releases the misfolded
protein into the lumen where it can fold, sequestered from the
cytoplasm
4. Another misfolded protein binds the opposite
side of the GroEL complex
Figure 6-87 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
Chaperone mediated protein folding
summary video/ tutorial
http://bcs.whfreeman.com/lodish5e/content/cat_010/03010-01.htm?v=chapter&i=03010.01&s=03000&n=00010&o=
MOLECULAR BIOLOGY – Protein structure & function
Unneeded and misfolded proteins are degraded by the
eukaryotic ‘proteasome’
20S core particle is formed from stacked
heptameric rings creating a hollow cavity
for safe protein digestion
Very large 2MDa protein nuclear &
cytoplasmic complex (26S)
19S regulatory
particle
20S core
particle
Structural/ regulatory
subunits
Proteolytically
active
subunits
How are condemned proteins targeted
to the proteasome ?
26S proteasome (cryo-electron microscopy)
MOLECULAR BIOLOGY – Protein structure & function
Protein are targetted to the protesome by ‘poly-ubiquitination’
‘Ubiquitin’ - 76 amino acid highly conserved polypeptide found in all cells that when attached to a
condemned protein in multiple copies targets it the proteasome (i.e. a destruction signal)
Ubiquitination of condemned
proteins requires three enzymes:
1) E1 ubiquitin activating enzyme
hydrolyses ATP to attaches itself to
and thus activate ubiquitin
2) E2 ubiquitin conjugating enzyme
recognises the E1-ubquitin complex
and transfers the complex to itself
3) E3 ubiquitin ligase enzyme binds
the condemned protein substrate
and an E2-ubiquitin complex thus
allowing E2 to transfer the ubiquitin
to the protein to be destroyed. This
process is repeated multiple times.
Poly-ubiquitinated proteins are then targeted to the proteasome where
ubiquitin is recognised by binding sites on the 19S particle and removed
for recycling. Energy from ATP hydrolysis unfolds and feeds the protein
into the catalytic core for destruction into amino acids and peptides
MOLECULAR BIOLOGY – Protein structure & function
Ubiquitin/ proteosome mediated protein
degradation summary video/ tutorial
http://www.sinauer.com/cooper5e/animation0802.html
MOLECULAR BIOLOGY – Protein structure & function
Protein folding timeline
Figure 6-88 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
Proteins not just targeted for destruction !
Specific sequences of amino acids can direct proteins to the correct sub-cellular location
for their function (e.g. the nucleus, mitochondria etc.)
e.g. ‘signal sequences’ targeting proteins for cell export
2) Translation is stalled and the SRP targets the ribosome
to a membrane ‘translocation complex’
1) a 20 amino acid
N-terminal
‘signal
sequence’ emerging
from the ribosome is
bound by the ‘signal
recognition particle
(SRP)’
3) SRP dissociation restarts translation
and the growing polypeptide chain is past
across the membrane ‘co-translationally’
Highly conserved between
eukaryotes and prokaryotes
(eukaryotic exported proteins pass across
the ER membrane rather than the plasma
membrane)
4) The signal sequence is enzymatically removed and the
protein folds
MOLECULAR BIOLOGY – Protein structure & function
Protein targeting e.g. signal recognition
sequences and cell export summary video/
tutorial
http://www.sinauer.com/cooper5e/animation1001.html
MOLECULAR BIOLOGY – Protein structure & function
Proteins perform many diverse functions
Information processing proteins
receptors, signalling
Structural proteins
Cytoskeleton
Extracellualr matrix
Enzymes
Mechanical proteins
actin, myosin
Binding proteins
transport, storage
Proteins are therefore subject to tight regulation to control these functions
MOLECULAR BIOLOGY – Protein structure & function
Enzymes comprise a large family of proteins
Enzymes and the reactions that they catalyse are central to regulating the activity
and function of other proteins i.e. they are important regulators
Table 3-1 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
Enzymes can modify proteins by the addition of molecular
moieties i.e. ‘post-translational modifications’
Although the genetic code specifies for the incorporation of only 20 amino acids into
proteins, these can be extensively modified to confer differing functionalities by:
Phosphorylation
Glycosylation
Methylation
N-acetylation
N-myristoylation
Deamination
S-prenylation
Sumoylation
S-pamitoylation
GPI-anchoring
Lipidation
Ubiquitination
S-Nitrosylation
Lipidation
MOLECULAR BIOLOGY – Protein structure & function
There exists huge potential for complex post-translational
regulation of protein function
e.g. multiple possible combinations of post-translational modification of the transcription factor p53.
Figure 3-81a Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
Post-translational protein modifications help increase the
possible variety in the products of a single gene
Such increased variety in the proteome allows for greater regulation of
protein function and output !
MOLECULAR BIOLOGY – Protein structure & function
e.g. phosphorylation status of an enzyme can dictate its activity
The interplay between the
kinase (adding phosphate) and
the phosphatase (removing
phosphate) regulates whether
enzyme ‘x’ is active or not
Figure 3-64 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
Glycogen metabolism in the liver is regulated by phosphorylation
A signal to stop storing glucose as glycogen and to start
mobilising it is processed through protein kinase A.
Phosphorylation of glycogen synthase
inactivates glycogen production
Whereas phosphorylation of
glycogen phoshorylase causes it’s
activation (via an intermediate
kinase) leading to glycogen
breakdown and glucose
production
Figure 3-73 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Protein structure & function
Various signals input into the establishment and hence readout of
this ‘code’
Figure 3-81c Molecular Biology of the Cell (© Garland Science 2008)