Transcript EF-Tu

Antecedentes históricos
Co-linearidad entre el ADN y la proteína
codificada por ese ADN.
•
Yanofsky, demostró que el orden de ciertas mutaciones en el gen
de la triptofano sintetasa era el mismo al de los cambios de
aminoácidos en la proteína
•
Crick y Brenner a partir de una larga colección de doble mutantes
de T4, que el código genético es leído en forma secuencial a partir
de un punto fijo
Estos experimentos sólo indican una correlación
El diccionario preciso del código genético
se determinó utilizando sistemas de
traducción in vitro derivado de células de
E.coli
Har Gobind Khorana
PREFERRED CODONS FOR SELECTED SPECIES
The following table lists the preferred codons in each species, along with
their usage as a percent of all codons for that amino acid.
Codons that differ from those preferred in man and rat are highlighted
Amino
acid
Human
Rat
E. coli
S. cerevisiae
S. frugiperda
Preferred
codon
GCC
% use
Preferred
codon
GCC
% use
38
Preferred
codon
GCT
% use
34
Preferred
codon
GCT
% use
41
Preferred
codon
GCC
% use
41
Arg
CGG
21
AGG
21
CGC
38
AGA
48
AGA
24
Asn
AAC
55
AAC
60
AAC
54
AAT
59
AAC
63
Asp
GAC
54
GAC
58
GAT
63
GAT
65
GAC
58
Cys
TGC
56
TGC
56
TGC
55
TGT
63
TGC
58
Gln
CAG
75
CAG
76
CAG
66
CAA
69
CAG
51
Glu
GAG
59
GAG
62
GAA
68
GAA
71
GAG
52
Gly
GGC
35
GGC
35
GGC
39
GGT
47
GGA
32
His
CAC
59
CAC
62
CAT
57
CAT
64
CAC
60
Ile
ATC
50
ATC
55
ATT
50
ATT
46
ATC
47
Leu
CTG
41
CTG
42
CTG
49
TTG
29
CTG
31
Lys
AAG
58
AAG
64
AAA
75
AAA
58
AAG
58
Met
ATG
100
ATG
100
ATG
100
ATG
100
ATG
100
Phe
TTC
56
TTC
60
TTT
57
TTT
59
TTC
65
Pro
CCC
33
CCC
32
CCG
51
CCA
41
CCT/CCA
29
Ser
AGC
24
AGC
25
AGC
26
TCT
27
TCC
20
Thr
ACC
37
ACC
38
ACC
42
ACT
35
ACT
32
Trp
TGG
100
TGG
100
TGG
100
TGG
100
TGG
100
Tyr
TAC
57
TAC
61
TAT
58
TAT
56
TAC
67
Val
GTG
48
GTG
48
GTG
36
GTT
39
GTG
39
Trm
TGA
51
TGA
50
TAA
62
TAA
48
TAA
64
Ala
37
“La molécula adaptadora”
A partir de la
secuencia de 300
tRNAs
La Hipótesis del Balanceo
(The Wobble Hypothesis)
Las células contienen diferentes
tRNAs que son específicos para
el mismo aminoácido
Muchos tRNAs unen a 2 o 3
codones
Activación de los aminoácidos
Al menos 20 aminoacil-tRNA sintetasas
La activación requiere ATP
La especificidad no es determinada por el
anticodón
Proof read antes de liberar el producto
X174
Iniciación
•
Se requiere un tRNA específico (tRNAmeti)
En E.coli una vez unida la metionina se formila
En ecuariotas tRNAmeti es específico pero no es formilado
•
El codón de iniciación (AUG)
En procariotas es localizado adyacente al elemento
Shine-Dalgarno
En ecuariotas es GENERALMENTE el primer AUG
encontrado por el ribosoma (A/G CCA/G CC AUG A/G)
Secuencia de reconocimiento de la iniciación
de la traducción
Small GTP-binding
proteins require
helper proteins, to
• facilitate
GDP/GTP
exchange, or
• promote GTP
hydrolysis.
G protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
G protein-GDP (inactive)
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 GTPbinding proteins
Initiation of protein synthesis in E. coli requires
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 aa-tRNA
fMet-tRNAfMet can bind only 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 fMet-tRNAfMet & helps
it to dock with the small ribosome subunit.
 As mRNA binds, IF-3 helps to correctly position the
complex such that the 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. This positions the 30S
ribosomal subunit in relation to the initiation codon.
 As the large ribosomal subunit 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.
Elongation cycle
Ribosome structure
and position of
factors & tRNAs
based on cryo-EM
with 3D image
reconstruction.
Diagram provided
by Dr. J. Frank,
Wadsworth Center,
NYS Dept. of Health.
Partial images on
subsequent slides are
derived from this.
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 EF1A)
• EF-Ts (EF1B)
• EF-G (EF2)
EF-Tu & EF-G are small GTP-binding proteins.
The sequence of events follows.
EF-Tu-GTP binds & delivers an
aminoacyl-tRNA to the A site
on the ribosome.
EF-Tu recognizes & binds all
aminoacyl-tRNAs with approx.
the same affinity, when each
tRNA is bonded to the correct
(cognate) amino acid.
tRNAs for different amino acids
have evolved to differ slightly
EF-Tu colored red
in structure, to compensate for
different binding affinities of amino acid side-chains, so the
aminoacyl-tRNAs all have similar affinity for EF-Tu.
The tRNA must have the correct anticodon to interact
with the mRNA codon positioned at the A site to form a
base pair of appropriate geometry.
Universally conserved bases of 16S rRNA interact with
and sense the configuration of the minor groove of the
short stretch of double helix formed from the first 2 base
pairs of the codon/anticodon complex.
A particular ribosomal conformation is stabilized by this
interaction, providing a mechanism for detecting whether
the correct tRNA has bound.
Proofreading in part involves release of the aminoacyltRNA prior to peptide bond formation, if the appropriate
ribosomal conformation is not generated by this
interaction.
EF-Tu-GTP
ribosome (GAP)
Pi
EF-Tu-GDP
The change in ribosomal conformational associated
with formation of a correct codon-anticodon complex
leads to altered positions of active site residues in the
bound EF-Tu, with activation of EF-Tu GTPase
activity.
The ribosome thus functions as GAP for EF-Tu.
When EF-Tu delivers an
aminoacyl-tRNA to the
ribosome, the tRNA initially
has a distorted conformation.
As GTP on EF-Tu is
hydrolyzed to GDP + Pi ,
EF-Tu undergoes a large
conformational change &
dissociates from the complex.
The tRNA conformation
relaxes, & the acceptor stem
is repositioned to promote
peptide bond formation.
This process is called accommodation.
EF-Tu colored red
It includes rotation of the
single-stranded 3' end of the
acceptor stem of the A-site
tRNA around an axis that
bisects the peptidyl transferase
center of the ribosomal large
subunit.
This positions the 3' end with
its attached amino acid in the
active site, near the 3' end of
the P-site tRNA, & adjacent to
the mouth of the tunnel
through which nascent polypeptides exit the ribosome.
PDB 1GIX
acceptor stems
of P-site &
A-site tRNAs
EF-Tu-GTP*
GDP
EF-Ts (GEF)
ribosome (GAP)
GTP
EF-Ts
functions as
GEF to
reactivate
EF-Tu.
Pi
EF-Tu-GDP**
*EF-Tu-GTP (conformation 1) binds &
delivers aa-tRNA to A site on ribosome.
**EF-Tu-GDP (conformation 2)
dissociates from complex.
Interaction with EF-Ts causes EF-Tu to release GDP.
Upon dissociation of EF-Ts, EF-Tu binds GTP, which is
present in the cytosol at higher concentration than GDP.
tRNA
P site
tRNA
A site
Transpeptidation
O
O
Adenine
Adenine O P O CH
(peptide bond
O P O CH
O
O
H
H
O
H
H
O
formation) involves
H
H
H
H
O
OH
O
OH
nucleophilic attack
O C
O C
of the amino N of
HC R
HC R
the amino acid
:NH
NH
linked to the 3'OH
of the terminal
O C
HC R
adenosine of the
NH
tRNA in the A site
on the carbonyl C of the amino acid (with attached nascent
polypeptide) in ester linkage to the tRNA in the P site.
2
2


2
3
+
The reaction is promoted by the geometry of the active site
consisting solely of residues of the 23S rRNA of the large
ribosomal subunit. No protein is found at the active site.
tRNA
P site
O
O
O
P
A site
tRNA
O CH2
O
H
O
O
H
H
O
H
OH
O
P
O
O CH2

H
O
C
HC
R
Adenine
O
H
H
O
H
OH
C
HC
R
:NH2
NH
O
Adenine
C
HC
R
NH3+
The 23S rRNA may be considered a "ribozyme."
As part of the reaction a proton (H+) is extracted from the
attacking amino N.
tRNA
P site
O
O
O
P
A site
tRNA
O CH2
O
H
Adenine
O
H
H
OH
H
OH
O
P
O CH2
O
H
O
Adenine
O
H
H
O
H
OH
C
HC
R
NH
O
C
HC
R
NH
O
C
HC
R
NH3+
This H+ is then donated to the hydroxyl of the tRNA in the
P site, as the ester linkage is cleaved.
tRNA
P site
O
O
O
P
O
O CH2

H
The nascent
polypeptide, one
residue longer,
is now linked to
the A-site tRNA.
A site
tRNA
Adenine
O
H
H
OH
H
OH
O
P
O
O CH2

H
O
O
H
H
O
H
OH
C
HC
R
NH
O
C
HC
R
NH
O
Adenine
C
HC
R
NH3+
tRNA grey,
EF-Tu red,
EF-G blue
The unloaded tRNA in the P site will shift to the E (exit)
site during translocation.
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.
Structural studies & molecular dynamics indicate that
EF-G-GTP binding in the vicinity of the A site causes a
ratchet-like motion of the small ribosomal subunit against
the large subunit.
large subunit
tRNA
EF-G
small subunit
mRNA
location
The tRNA with attached nascent polypeptide is pushed
from the A site to the P site.
Unloaded tRNA that was in the P site shifts to the E site.
Since tRNAs are linked to mRNA by codon-anticodon base
pairing, the mRNA moves relative to the ribosome.
En E.coli hay 2 RFs, en eucariotas 1 RF
UAA y UAG son reconocidos por RF-1
UAA y UGA son reconocidos por RF-2
En eucariotes
El primer paso es la formación del complejo GTP – eIF-2
eIF-GTP se une a tRNA i
Se une a la subunidad 40S (complejo 43S)
Luego se estabiliza con eIF-3 y eIF-1
El cap se une a eIF-4F: compuesto por 4E, A y G
4E se une a cap
4A une ATP y tiene actividad RNA helicasa
4G ayuda a la unión al complejo 43 S
Hipótesis
eIF-4E es el IF en menor nivel (determinante)
Se regula a nivel de
 Transcripción
 Modificación (fosforilación )
 Inhibición
Regulation of Step 1
 1. Phosphorylation of the eIF4E Binding Proteins,
the 4E-BPs.
 2. Binding of PolyAdenylate Binding Protein (PABP)
to eIF4G.
Why?
Because this circularizes
the polysome, and allows
ribosomal subunits to start
new ribosomes.
MAPK-Dependent Phosphorylation of eIF4E
Is Mediated by the eIF4G Associated Kinase Mnk
vía MAPK/ERK o vía MAPK (de las siglas
en inglés Mitogen-activated protein kinases
Phosphorylation
of eIF4E allows it
to detach from the
cap and recycle
Binding of PolyAdenylate Binding Protein (PABP) to
eIF4G
• The translational control of maternally inherited mRNAs is a key
feature of early animal development
• mRNAs are synthesized and stored (i.e., masked) during the
protracted period of oogenesis and are translated only in response
to subsequent exogenous cues
• the oocytes of Xenopus laevis contain masked mRNAs that are
activated during the cell’s reentry into meiosis(oocyte maturation), a
process that is stimulated by the interaction of progesterone with a
surface-associated receptor
In case of mRNAs with a CPE sequence in the 3’ end, the poly(A)
tail also serves to disrupt the binding of maskin, a CPEB-binding
protein to eIF4E. This makes eIF4E available to start building the
cap-binding complex
Control de la síntesis de Hemo por
traducción
Regulación de la iniciación
Control de la síntesis de Hemo por
traducción
• Protein synthesis in intact reticulocytes and their lysates is
dependent on the availability of heme. In heme deficiency,
protein synthesis is inhibited with increased phosphorylation of
the a subunit of eIF2.
• The phosphorylation of eIF2 in heme deficiency is the result of
the activation of heme-regulated inhibitor (HRI), which is a
heme-regulated eIF2 kinase
GTP
eIF-2
GEF
(eIF-2B)
Bajo hemo
GDP
eIF-2
HRI
HRI
inactivo
activo
Alto hemo
GDP
eIF-2
P
GEF GDP
(eIF-2B)
eIF-2
P
GEF
(eIF-2B)
Elongación
Amino acid-containing tRNA
molecules (aminoacyl-tRNAs, aatRNA) are picked up by
elongation factor eEF-1 in the
presence of GTP.
The empty tRNA is displaced
from the P-site to the E-site as the
peptidyl tRNA is translocated
from the A-site to the P-site. The
process is facilitated by
elongation factor eEF-2 and
GTP.
Terminación
 Releasing Factors (eRFs) are involved in termination.
 eRF1 structurally mimics tRNA that is bound to eEF1a •
GTP. eRF1 fits into the ribosomal A-site, where it
recognizes the stop codon. It then releases the completed
polypeptide by catalyzing a nucleophilic attack on the ester
bond between the peptide and the P-site tRNA.
 The catalytic activity of eRF1 is stimulated by the GTPbound form of another relasing factor, eRF3.
Domain 2 on eRF1 Recognizes the Stop Codon
Domain 3 Catalyzes the Hydrolysis of the Completed Peptide from the P-Site tRNA
Se une a la subunidad 30S del ribosoma
bacteriano bloqueando la fijación del aminoaciltRNA al sitio aceptor (A) del complejo formado
por el mRNA y la subunidad 50S del ribosoma
La kanamicina afecta a la subunidad 30S
de los ribosomas y causa una mutación
con cambio previniendo la traducción del
ARN.
Aminoacil- tARN, en vez de leer el codón
CUA —como sería el caso en la secuencia
CUAG— se lee el codón UAG (codón ambar
de terminación)
Translational Frameshift
UGA C
GAC
UGA
RF2
Pausa
Frameshift
(UGAC se lee
como GAC y
sigue +1)
RF2
Unión de RF2
terminación de la traducción
Determinación de la dirección de síntesis de las proteínas
EXPERIMENTO DE DINTZIS
Ingram’s fingerprinting technique was performed by purifying
hemoglobin from red blood cells, fragmenting hemoglobin protein
into peptides with the enzyme trypsin, separating the fragments
(based on their respective charges) by electrophoresis, and staining
his results. In this way he produced a “fingerprint” of the protein, as
the different amino acids would migrate to different locations on the
electrophoretic gradient based on their charges.
Dintzis added 14C-labeled amino acids to mature reticulocytes, which
are always involved in synthesizing hemoglobin.
¿Qué resultados se obtienen?
¿Qué se grafica?