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MB 207 – Molecular Cell Biology
From RNA to Protein
• Translation
• Protein
Overall view of Protein Synthesis
The ribosome
(ribosomes read messenger RNA and direct the synthesis of
the encoded protein)
• The cellular factory responsible for synthesizing proteins
• Consists of various rRNAs and about 50 different proteins (ribosomal proteins): 2/3
RNAs & 1/3 proteins
• RNAs instead of proteins with the catalytic activity, proteins are there as to stabilize
the RNA core
• Assembled in the nucleolus
• Inactive state: exists as two subunits: a large subunit & a small subunit
• When the small subunit encounters an
mRNA, the process of translation of the
mRNA to protein begins.
• Contain 4 binding sites:
1 mRNA binding site &
3 tRNA binding sites:
 A (aminoacyl)
 P (Peptidyl)
 E (Exit) site
Comparison procaryotic and eucaryotic ribosome structures
(both have similar structure and function)
S= rate of sedimentation in an ultracentrifuge
Ribosome subunits
Prokaryotes
Eukaryotes
Small subunits
30S
40S
Large subunits
50S
60S
Whole ribosome 70S
80S
Svedberg (S): a unit of sedimentation
velocity (sedimentation is an ultracentrifuge
depends on both the mass and shape of a
molecule
Charging the tRNA
•
tRNA is the translator between mRNA and protein (transfer genetic information to
aa sequence)
• tRNA has a specific anticodon and acceptor site
• tRNA has a specific charger protein - aminoacyl tRNA synthetases
 can only bind to that particular tRNA and attach the correct amino acid to the
acceptor site
 Energy to make this bond comes from ATP
The genetic code is translated by means of the two adaptors that act one after another:
1) aminoacyl-tRNA synthetase (couples an aa to it’s corresponding tRNA)
2) tRNA molecule (anticodons forms bp with codon on the mRNA)
anticodon
codon
Translation
• The process that uses the base sequence in mRNA to synthesize a
polypeptide with "complementary" amino acid sequence
• The information in mRNA is always read from the 5' to the 3'
direction. The polypeptide is synthesized from its amino terminus to
its carboxyl terminus.
RNA: 5'----------------------------------3'
polypeptide: H2N-----------------------------COOH
• The translation process occurs at the ribosome in the cytoplasm
• Involves the three major classes of RNA: mRNA, tRNA, and rRNA
as well as free amino acids, free energy, and several non-ribosomal
protein factors
The ribosomal subunit cycle during protein synthesis
Initiation factors
30S subunits
with initiation
factors
Initiation
Separate
subunits
Pool of free
ribosomes
(Ribosomal subunits
assemble and
disassemble during each
round of protein
synthesis in the
ribosome cycle)
Elongation
Termination
Initiation requires free ribosome subunits. When ribosomes are released at termination,
they dissociate to generate free subunits. Initiation factors are present only on
dissociated 30S subunits. When subunits reassociate to give a functional ribosome at
initiation, they release the factors.
Translation initiation requires:
Ribosome brought to mRNA
Ribosome properly aligned over start codon
P site of ribosome containing charged tRNA
The initiation process differs significantly in prokaryotes and eukaryotes
Initiation is aided by translation initiation factors
Translation initiation
Prokaryotes Eukaryotes
Function
IF1
eIFA
Blocks ribosome A site
IF2
eIF, eIF5b
Facilitate initiator tRNA
binding to the P site of 40S
subunit
IF3
eIF3
Prevents ribosomal subunit
association
Prepare mRNA template for
ribosome binding
eIF, eIF4A,
eIF4E,
eIF4G,
eIF4B
Bacterial mRNA are often polycistronic – encode several different proteins
Eucaryotes mRNA only encode 1 single protein
Translation initiation in Prokaryotes
IF1, IF2 and IF3 bind 30S subunits (IF3, then IF2-GTP and IF1)
IF1 prevents tRNAs from entering A site
IF2 binds IF1 and guides fMet-tRNA to P site
IF3 prevents association of large subunit
Shine-Dalgarno sequence base pairs with 16S rRNA on ribosome
•mRNA start codon and fMet-tRNA anticodon are aligned to start translation
•Base-pairing of mRNA start codon and fMet-tRNA initiates reaction cascade to form
70S initiation complex:
IF3 dissociates
Large subuntis binds
GTP is hydrolyzed by IF2
IF2 and IF1 dissociate
•Protein synthesis begins
Initiation of Translation
• The start signal for translation is the codon ATG (AUG), which codes for
methionine (met) (formyl-met for prokaryotes) – all newly synthesized
protein has a met at the N-terminal
• A tRNA charged with met is required to bind to the translation start signal
• An complex consists of the small ribosomal unit, an initiation factor
(eIFs) and the tRNAmet is formed
• The complex bind to the 5’ end of an mRNA (recognize the 5‘ Cap for
eukaryotes or Shine-Dalgarno sequence for prokaryotes) and move
forward along the mRNA to search for the initiator codon AUG
- Scanning requires energy - ATP hydrolysis
- It is not always the first AUG that is recognized as a start codon,
sequences around first AUG might reduce efficiency of initiation so
scanning continues to next AUG.
• Initiation factors dissociate with GTP hydrolysis, and the large ribosomal
subunit associates with the complex, the initiator tRNA bind at P site
• Protein synthesis is ready to begin
The initiation phase of protein synthesis in eukaryotes
43S pre-initiation
complex formed by:
•40S subunit
•Initiation factors eIFA,
eIF3, eIF5B-GTP and eIFMet-tRNA-GTP
eFI4F complex (E,G and
A) binds to mRNA
43S pre-initiation
complex brought to mRNA
by eIF4 family of
translation initiation factors
Helicase activity of eIF4
family helps 40S subunit
seat on the start codon
eIF2 and eIF3 are
released from the complex
60S
subunit
binds 40S
subunit and
initiation
factors are
released
Ribosome
is now
ready to
begin
protein
synthesis
Elongation requires:
Placement of charged tRNA in the A
site
Peptide bond formation
Translocation
Elongation factors EF-Tu, EF-G and
EF-Ts
Elongation of the New Protein
• A next tRNA carrying an other amino acid is attracted and pairs with the next
codon at the A site, the peptide bond is catalysed by a ribosomal protein
(peptidyl-transferase) associated with the large ribosomal subunit.
• The result is a transfer of the N-methionine to the second tRNA in the A site
which carries now a dipeptide and the initiator tRNA being uncharged in the P
site.
• Formation of each peptide bond is energetically favorable because the growing
C-terminus has been activated by the covalent attachment of a tRNA molecule
The incorporation of an amino acid into a protein
Elongation of the New Protein
• The first tRNA is now released (move to E site) and the ribosome shifts so
that a tRNA carrying two amino acids is now in the P site, and leaves the A
site unoccupied but with the third codon exposed.
• Translocation:
– Ribosome moves to the next codon
– Empty tRNA is ejected and the peptidyl-tRNA is shifted from the A site
to the P site
– New aminoacyl-tRNA is allowed to enter within the A site
– Translocation is catalyzed by the elongation factor EF-G.
The G indicates that this factor uses the energy gained from the
hydrolysis of GTP for translocation to occur.
– Finally a third tRNA is escorted by EF-Tu to the A site and the
anticodon base pairs specifically with the codon.
– This binding triggers the release of the initiator tRNA from the E site
and allows the complex to be ready for another round of peptide bond
formation, translocation and codon-anticodon base pairing
– And so on…..
View of the translation cycle
The aminoacyltRNA is escorted
to the A site by the
elongation factor
EF-Tu
The peptidyl
transferase
reaction transfer
the amino acid
from the P site
onto the
aminoacyl-tRNA
in the A site
Incorrectly base paired tRNAs
preferentially dissociate
During
translocation:
•Ribosome
moves one
codon down
mRNA
•tRNA with
growing protein
chain moves into
P site
•Spent tRNA
moves into E site
•Translocation
requires EF-G
Translating an mRNA molecule
Step 1 An aminoacyl-tRNA bind to a vacant A-site
on the ribosome.
Step 2 A new peptide bond is form
Step 3 The mRNA moves a distance of 3 nts through the
small –subunit chain, ejecting the spent tRNA
molecule and ‘resetting’ the ribosome so that the
next incoming aminoacyl-tRNA molecule can bind
The 3 steps cycle is
repeated over and
over again during
protein synthesis.
Termination
Stop codon signals termination: UAG, UGA, UAA
Release factors (RF) accomplish termination
Termination is similar in prokaryotes and eukaryotes
Prokaryotes
•Class I
Eukaryotes
•RF1 recognizes stop codon eRF1 recognizes all
UAG
stop codons
•RF2 recognizes stop codon
UGA
•RF1 & 2 recognizestop
codon UAA
•Class II •RF3
eRF3
Termination of the
Protein Synthesis
• When the ribosome reaches a stop
codon, no aminoacyl tRNA binds to
the empty A site. This is the
ribosomes signal to break into its
large and small subunits, releasing
the new protein and the mRNA.
• Stop codons are triplets which are
not recognized by any tRNA (UAA,
UAG, UGA), but by a protein
releasing factor (RF1 or RF2 in
prokaryotes, eRF in eukaryotes).
• The factor R binds to the A site
forcing the peptidyl transferase to
catalyze the addition of water to the
peptidyl-tRNA and causes the
release of the polypeptide chain
•This release in
turn causes the
complex to
dissociate and
mRNA, tRNA
and ribosomal
subunits are
freed.
The final phase of
protein synthesis
Polyribosome
• A single mRNA molecule is not
only translated once
– As soon as the ribosome
has moved away from the
initiation site, another round
of initiation can begin
– A single mRNA is often
transcribed by many
ribosomes at the same time,
usually 100 to 200 bases
apart from each other
• A group of ribosomes on the
same mRNA is called a
polyribosome or polysome
• Many proteins can be made in a
given time
Protein folding
• Polypeptide chain acquires its secondary and tertiary structure
as it emerges from a ribosome. The N-terminal domain folds
first, while the C-terminal domain is still being synthesized.
• The protein has not yet achieved its final conformation by the
time it is released from the ribosome.
• Mechanisms that monitor protein quality after protein synthesis:
1) Correctly fold and assemble protein will be left alone
2) Incompletely folded proteins were refolded, with the help
from molecular chaperones: e.g. hsp70, hsp60-like proteins
3) Incompletely folded proteins that can not be refold will be
digested by proteosomes
4) Combination of all of these processes is needed to prevent
massive protein aggregation in a cell
Action of chaperones during translation
Chaperones bind to the amino (N) terminus of the growing polypeptide
chain, stabilizing it in an unfolded configuration until synthesis of the
polypeptide is completed.
Protein folding
The co-translational folding of a protein:
- A growing polypeptide chain is shown acquiring its secondary & tertiary structure as it emerges
from a ribosome.
The hsp70 family of molecular
chaperones:
Recognize a small stretch of hydrophobic
amino acids on a protein’s surface.
Hsp70 (together with hsp40) binds to it’s
target protein and hydrolyzes a molecule
of ATP to ADP, undergoing a conformational change result in hsp70 clamping
tightly to target. Hsp 70 then dissociate
induced by rapid
hsp70
re-binding of ATP.
machinery
hsp70
machinery
Repeated cycles of hsp
protein binding & release
help the target protein
to refold.
The cellular mechanisms that monitor protein quality after protein
synthesis:
• Combination of processes needed to prevent massive aggregation in a cell,
which can occur when many hydrophobic regions on protein clump together
and precipitate the entire mass.
Protein
aggregate
Newly synthesized
protein
Hsp70, 60 & 40
Correctly folded
without help
Increasing time
Correctly folded
with help from
chaperone
Incompletely folded forms
– digested in proteosome
Different levels of protein structure
• Proteins come in a wide variety of shapes, and generally 50
to 2000 amino acids long
 Primary structure: Sequence of amino acids along the
core of the polypeptide chain.
 Secondary structure: Folding of the polypeptide into most
energetically favorable conformation resulting from
various non-covalent bonds that form between one part of
the chain and another.
 Tertiary structure: The full 3-D organization of a
polypeptide chain.
 Quaternary structure: The highest level of organization,
recognized in protein, formed as a complex of more than
one polypeptide
Polar and Nonpolar Amino acids
Primary
structure
Secondary Structure
• Four different noncovalent bonds: ionic bond, H-bond, Van der Waals
attraction & hydrophobic/hydrophilic attraction
Folded conformation in
aqueous environment
Unfolded polypeptide
Secondary Structure
• Two most common types: -helix & -sheet
• -helices are held together by H-bond between the N-H and C=O groups
of the polypeptide backbone 4 aa away
– Form a regular helix with a complete turn of 3.6 aa
Secondary Structure
• -sheets are held together by H-bond between the N-H in the peptide
bond on one strand and the C=O of a peptide bond on another sheet
strand
– Produce a very rigid sheet structure
– parallel vs anti-parallel
-sheet
anti-parallel
parallel
Tertiary Structure
• Besides the 4 non-covalent bonds, one of the important features is
the formation of disulfide bonds S-S bonds (between cysteines)
cysteine
oxidants
reductants
Interchain
disulfide
bond
Intrachain
disulfide
bond
Tertiary Structure
• Protein domain: a substructure produced by any part of a
polypeptide chain that fold independently into a compact,
stable structure with a specific function, i.e. catalytic domain
SH3 domain
SH2 domain
Small kinase
domain
Large kinase domain
Quaternary structure
• Overall 3D structure assumed by the multimeric protein
• Aggregates of more than one polypeptide chain
• Individual polypeptide chains that make up multimeric proteins are often
called protein subunits
4o structure of Hemoglobin
Types of protein based on structure:
Globular & Fibrous
Globular:
– Tightly folded polypeptide chains, having a much more compact
structure.
– Globular proteins include most enzymes and most of the proteins
involved in gene expression and regulation, e.g. hemoglobin,
deoxyribonucleases, cytochrome c
DNAse
Cytochrome c
Types of protein based on structure
• Fibrous:
– Elongated structures, with the polypeptide chains
arranged in long strands (parallel strands along a
single axis).
– Major structural components of the cell or tissue
– Examples: collagen (tendon, cartilage, and bone), elastin (skin),
tubulin and actin (cell shape, motility, muscle movement)
Collagen
triple helix
Assembly of proteins
• Proteins molecules often serve as a subunits for the assembly of large
structure
• Benefits:
Free subunits
– A large structure built from
repeating subunits requires
smaller amount of genetic
information
– Both assembly and
disassembly can be readily
controlled, reversible
processes
– Errors in the synthesis of
the structure can be more
easily avoided, since
correction mechanisms can
operate during the course of
assembly to exclude
malformed subunits
Assembled structures
dimer
helix
ring
Proteins
• Many proteins have non-peptide components such as
carbohydrate moieties (glycoproteins), metal groups
(metalloproteins), lipids (lipoproteins).
• Function of proteins
– structure: hair, fingernails.
– transport: hemoglobin.
– information: protein hormones.
– catalysis: enzymes.
– locomotion: muscles.
• Protein family: a group of proteins with members having
similar
- amino acids sequence
- three-dimensional (3-D) structure &
- function
(eg. serine proteases)