Transcript No Slide Title

• There are many steps
to produce a protein in
a eukaryotic cell
• Each step is a point of
regulation to
determine the
efficiency of gene
expression
Translation
• Initiation
• Elongation
• Termination
Factors involved in initiation, elongation, &
termination of protein synthesis.
Many of these factors are GTP-binding
proteins, & other proteins that control GDP/GTP
exchange or GTPase activity of these GTPbinding proteins.
Heterotrimeric G-proteins.
 A GTP-binding protein has a different conformation
depending on whether it has bound to it GTP or GDP.
 Usually GTP binding induces the active conformation.
 Hydrolysis of the bound GTP to GDP + Pi converts
the protein to the inactive conformation.
 Reactivation occurs by release of bound GDP in
exchange for GTP.
Small GTP-binding
G protein-GTP (active)
proteins require
GDP
helper proteins, to
• facilitate
GEF
GAP
GDP/GTP
GTP
Pi
exchange, or
• promote GTP
G protein-GDP (inactive)
hydrolysis.
A guanine nucleotide exchange factor (GEF) induces a
conformational change that makes the nucleotide-binding
site of a GTP-binding protein more accessible to the
aqueous intracellular milieu, where [GTP]  [GDP].
Thus a GEF causes a GTP-binding protein to release
GDP & bind GTP (GDP/GTP exchange).
A GTPase
activating
protein (GAP)
causes a GTPbinding protein
to hydrolyze its
bound GTP to
GDP + Pi.
G protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
G protein-GDP (inactive)
The active site for GTP hydrolysis is on the GTP-binding
protein, although a GAP may contribute an essential active
site residue.
GEFs & GAPs may be separately regulated.
Unique GEFs and GAPs interact with different
GTP-binding proteins
Members of the family of small GTP-binding proteins
have diverse functions.
In some cases, the difference in conformation, with
substitution of GDP for GTP allows a GTP-binding
protein to serve as a "switch".
In other cases the conformational change may serve a
mechanical role or alter the ability of the protein to
bind to membranes.
Roles of some small GTP-binding proteins:
 IF-2, EF-Tu, EF-G, & RF-3: Protein synthesis
initiation, elongation, & release factors.
 Ras: Growth factor signal cascades.
 Rab: Membrane vesicle targeting & fusion.
 ARF: Vesicle budding by formation of coatomer coats.
 Ran: Transport of proteins into & out of the nucleus.
 Rho: Regulation of the actin cytoskeleton.
Translation initiation
Eukaryotes v. Prokaryotes
Initiation of protein synthesis in E. coli requires
participation of initiation factors IF-1, IF-2, & IF-3.
 IF-3 binds to the 30S ribosomal subunit, freeing it
from its complex with the 50S subunit.
 IF-1 assists binding of IF-3 to the 30S ribosomal
subunit.
IF-1 also occludes the A site of the small ribosomal
subunit, helping insure that the initiation tRNAfMet
will end up in the P site & that no other aa-tRNA can
bind in the A site during initiation.
 IF-2 is a small GTP-binding protein.
IF-2-GTP binds the initiator tRNAfMet & helps it to
dock with the small ribosome subunit.
 As mRNA binds, IF-3 helps to correctly position the
complex such that tRNAfMet interacts via base pairing
with the mRNA initiation codon (AUG).
A region of mRNA upstream of the initiation codon,
the Shine-Dalgarno sequence, base pairs with the
3' end of the 16S rRNA, helping to position the 30S
ribosomal subunit in relation to the initiation codon.
 The large ribosomal subunit then joins the complex.
GTP on IF-2 is hydrolyzed, leading to dissociation of
IF-2-GDP and dissociation of IF-1.
A domain of the large ribosomal subunit serves as
GAP (GTPase activating protein) for IF-2.
 Once the two ribosomal subunits come together, the
mRNA is threaded through a curved channel that
wraps around the "neck" region of the small subunit.
Initiation
Shine Delgarno Sites
initiation of translation in eukaryotes
1. charging of the tRNAs is the same
2. Association of the translation machinery
- terminology difference initiation factors called eIFs rather than IFs
- charged tRNA is delivered by eIF2-GTP (which is hydrolized to GDP
to provide energy)
- eukaryotic mRNA is capped, this is recognized by an additional protein
eIF-4E
3. identification of initiator codon
- ribosome tRNA complex scans for first AUG and stops there
- directed by the eIF-4E on the CAP site rather than the Shine-Delgarno
site
4. completion of initiation- same
Translation Initiation - Eukaryotes
Controlling Initiation
• Eukaryotic mRNA has cap on 5’ end
• Bacteria have no “5’ end marker”
– Translation is coupled to transcription
– Ribosomes bind to RNA as it is made
• Where to start reading mRNA?
– Shine Delgarno sequence in bacteria, Kozak
sequence in eukaryotes
– Adjacent to the first codon
The AUG start codon is recognized by
methionyl-tRNAiMet
Translation elongation
Elongation cycle
Ribosome structure
and position of
factors & tRNAs
based on cryo-EM
with 3D image
reconstruction.
Colors: large ribosome subunit, cyan; small subunit, pale yellow;
EF-Tu, red; EF-G, blue. tRNAs, gray, magenta, green, yellow, brown.
Elongation requires participation of elongation factors
• EF-Tu (also called EF-1A)
• EF-Ts (EF-1B)
• EF-G (EF-2)
EF-Tu & EF-G are small GTP-binding proteins.
The sequence of events follows.
EF-Tu-GTP binds and delivers an aminoacyl-tRNA to
the A site on the ribosome.
The loaded tRNA must have the correct anticodon to base
pair with the mRNA codon positioned at the A site.
tRNA binding causes a conformational change in the
small ribosomal subunit that causes universally conserved
bases of 16S rRNA to interact closely with the minor
groove of the first two base pairs of the codon/anticodon
complex, helping insure that only the correct tRNA binds.
Proofreading in part involves release of the aa-tRNA
prior to peptide bond formation if a particular ribosomal
conformation is not stabilized by this interaction.
As the aa-tRNA is
delivered by EF-Tu to
the A site on the ribosome,
GTP on EF-Tu is
hydrolyzed to GDP + Pi.
A domain of the ribosome
serves as GAP for EF-Tu.
This function depends
on codon-anticodon
recognition correctly
positioning the aa-tRNA in
relation to the large
ribosomal subunit.
EF-Tu colored red
A large conformational
change in EF-Tu, when
GTP  GDP + Pi,
promotes dissociation
of EF-Tu.
Release of EF-Tu leads
to repositioning of the
aa-tRNA to promote
peptide bond formation.
EF-Tu colored red
EF-Tu-GTP*
GDP
EF-Ts (GEF)
ribosome (GAP)
GTP
Pi
EF-Tu-GDP**
EF-Ts functions
as GEF to
reactivate EF-Tu.
*EF-Tu-GTP (conformation 1) binds &
delivers aa-tRNA to A site on ribosome.
**EF-Tu-GDP (conformation 2)
dissociates from complex.
EF-Ts induces EF-Tu to release bound GDP & bind GTP.
EF-Ts dissociates from EF-Tu when EF-Tu changes its
conformation, upon binding GTP.
Transpeptidation (peptide bond formation) involves
acid/base catalysis by a universally conserved adenosine
of the 23S rRNA of the large ribosomal subunit.
No protein is found adjacent to the active site adenosine.
(Recall Chime exercise on the large ribosomal subunit.)
The 23S rRNA may be considered a "ribozyme.“
The amino N of the amino acid linked to the 2' or 3' OH
of the terminal adenosine of tRNA in the A site reacts
with the carbonyl C of the amino acid (with attached
nascent polypeptide) linked to the tRNA in the P site.
tRNA
The nascent
polypeptide, one
residue longer, is
now linked to the
tRNA in the A site.
P site
O
O
O
P
O
A site
tRNA
O CH2

H
Adenine
O
H
H
OH
H
OH
O
P
O
O CH2

H
O
Adenine
O
H
H
O
H
OH
C
HC
R
However, peptide
NH
bond formation is
O C
associated with
HC R
rotation of the
NH
acceptor stem of
the A site tRNA, so
that the nascent
O C
polypeptide is
HC R
positioned to feed
NH3+
via the P site into
the tunnel in the large subunit. The unloaded tRNA in the P site will
shift to an exit (E) site during translocation.
tRNA grey, EF-Tu red, EF-G blue
Translocation of the ribosome relative to mRNA
involves the GTP-binding protein EF-G.
The size & shape of EF-G are comparable to that of the
complex of EF-Tu with an aa-tRNA.
large subunit
tRNA
EF-G
small subunit
mRNA
location
EF-G-GTP binds in the vicinity of the A site.
EF-G-GTP binding may push the tRNA with attached
nascent polypeptide from the A site to the P site.
Unloaded tRNA that was in the P site shifts to an exit site.
Since tRNAs are linked to mRNA by codon-anticodon base
pairing, the mRNA would move relative to the ribosome.
Additionally, it has been postulated that translocation is
spontaneous after peptide bond formation because:
• the deacylated tRNA in the P site has a higher
affinity for the E site, &
• the peptidyl-tRNA in the A site has a higher affinity
for the P site.
Interaction with the ribosome, which acts as GAP
(GTPase activating protein) for EF-G, causes EF-G to
hydrolyze its bound GTP to GDP + Pi.
EF-G-GDP then dissociates from the ribosome.
A domain of EF-G functions as its own GEF (guanine
nucleotide exchange factor) to regenerate EF-G-GTP.
Bacterial Elongation
• Elongation factor Tu uses GTP hydrolysis to
help bring tRNA to A site
• Elongation factor Ts helps restore fresh
GTP to EF-Tu
• Elongation factor G uses GTP hydrolysis to
help move tRNA from A to P site
Eukaryotic Elongation
• One factor (eEF-1) does job of EF-Tu and
EF-Ts
• eEF-2 does job of EF-G
• As with bacteria, the elongation factors
work within the ribosome
Translation termination
Eukaryotes v. Prokaryotes
Termination
• Three codons lack complementary tRNAs
• Recognized by “release factors”
–
–
–
–
Three proteins in bacteria
Two can bind to “stop codon”
Third helps with interaction, uses GTP
Eukaryotes have one release factor
• Induce the breakage of tRNA-amino acid
bond of tRNA in P site
 RF-1 & RF-2 recognize & bind to STOP codons.
One or the other binds when a stop codon is reached.
 RF-3-GTP facilitates binding of RF-1 or RF-2 to the
ribosome.
 Once release factors occupy the A site, Peptidyl
Transferase catalyzes transfer of the peptidyl group to
water (hydrolysis). The uncharged tRNA is released.
 Hydrolysis of GTP on RF-3 causes a conformational
change that results in dissociation of the release
factors. The ribosome dissociates from mRNA.
 IF-3, assisted by IF-1, promotes dissociation of the
two ribosomal subunits for another round of initiation.
termination, directed by the STOP codon
A
3’
AUG GCC UUU
5’
EF-G-GDP
EF-G-GTP
Release factor
A
5’
AUG GCC UAA
A: (aminoacyl) site
P: (peptidyl) site
E: (exit) site
3’
Polyribosomes. these structures are
formed by the presence of several
ribosomes working sequentially on a
single mRNA
The ribosome by SEM
Functional concept of the ribosome
Antibiotic Action
Puromycin Mechanism
Cap-dependent vs.
cap-independent translation initiation
Cap-Dependent
Cap-Independent
43S particle recruitment strategies