Antecedentes históricosAntecedentes 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 correlaciónEstos experimentos sólo indican una correlación

El diccionario preciso del código genético El diccionario preciso del código genético se determinó utilizando sistemas de se determinó utilizando sistemas de traducción in vitro derivado de células de traducción in vitro derivado de células de E.coliE.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

% use Preferred codon

% use Preferred codon

% use Preferred codon

% use Preferred codon

% use

Ala GCC 41 GCC 41 GCC 34 GCT 38 GCT 37

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

 

“La molécula adaptadora”

A partir de la A partir de la secuencia de 300 secuencia de 300

tRNAstRNAs

La Hipótesis del Balanceo La Hipótesis del Balanceo (The Wobble Hypothesis)(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 aminoácidosActivación de los aminoácidos

Al menos 20 aminoacil-tRNA sintetasas

La activación requiere ATP

Proof read antes de liberar el producto

La especificidad no es determinada por el anticodón

X174

Iniciación 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-DalgarnoShine-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 Secuencia de reconocimiento de la iniciación de la traducciónde la traducción

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).

G protein-GTP (active)

GDP

GEF GAP

GTP Pi

G protein-GDP (inactive)

Small GTP-binding proteins require helper proteins, to • facilitate

GDP/GTP exchange, or

• promote GTP hydrolysis.

G protein-GTP (active)

GDP

GEF GAP

GTP 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

A GTPase activating protein (GAP) causes a GTP-binding protein to hydrolyze its bound GTP to GDP + Pi.

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.

Initiation of protein synthesis in E. coli requires initiation factors IF-1, IF-2, & IF-3.

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.

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.

Colors: large ribosome subunit, cyan; small subunit, pale yellow; EF-Tu, red; EF-G, blue. tRNAs, gray, magenta, green, yellow, brown.

Elongation cycleRibosome 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.

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 in structure, to compensate for different binding affinities of amino acid side-chains, so the aminoacyl-tRNAs all have similar affinity for EF-Tu.

EF-Tu colored red

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 aminoacyl-tRNA prior to peptide bond formation, if the appropriate ribosomal conformation is not generated by this interaction.

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.

EF-Tu-GTP ribosome (GAP) Pi EF-Tu-GDP

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 poly-peptides exit the ribosome.

acceptor stems of P-site & A-site tRNAs

PDB 1GIX

EF-Tu-GTP*

GDP

EF-Ts (GEF) ribosome (GAP)

GTP 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.

EF-Ts functions as GEF to reactivate EF-Tu.

Transpeptidation (peptide bond formation) involves nucleophilic attack of the amino N of the amino acid linked to the 3'OH of the terminal adenosine of the 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.

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.

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH

R

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH2

R

C

HC

NH3+

R

O

O

O

:

P site A site

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH

R

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH2

R

C

HC

NH3+

R

O

O

O

:

P site A site

The 23S rRNA may be considered a "ribozyme." 

As part of the reaction a proton (H+) is extracted from the attacking amino N.

This H+ is then donated to the hydroxyl of the tRNA in the P site, as the ester linkage is cleaved.

O

OHOH

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH

R

O

C

HC

NH

R

C

HC

NH3+

R

O

O

P site A site

The nascent polypeptide, one residue longer, is now linked to the A-site tRNA.

O

OHOH

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH

R

O

C

HC

NH

R

C

HC

NH3+

R

O

O

P site A site

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.

tRNA grey, EF-Tu red, EF-G blue

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.

small subunit

large subunit

mRNAlocation

EF-G

tRNA

En E.coli hay 2 RFs, en eucariotas 1 RFEn 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 GEl 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

1. Phosphorylation of the eIF4E Binding Proteins, the 4E-BPs.

2. Binding of PolyAdenylate Binding Protein (PABP) to eIF4G.

Regulation of Step 1

Why?

Because this circularizes the polysome, and allows ribosomal subunits to start new ribosomes.

MAPK-Dependent Phosphorylation of eIF4EIs Mediated by the eIF4G Associated Kinase Mnk

Phosphorylation of eIF4E allows it to detach from the cap and recycle

vía MAPK/ERK o vía MAPK (de las siglas en inglés Mitogen-activated protein kinases

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

• 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 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

Control de la síntesis de Hemo por traducción

eIF-2GTP

eIF-2GDP

GEF(eIF-2B)

eIF-2GDP

P

eIF-2GDP

P

GEF(eIF-2B)

GEF(eIF-2B)

HRI

inactivo

HRI

activo

Alto hemo

Bajo hemo

Elongación

Amino acid-containing tRNA molecules (aminoacyl-tRNAs, aa-tRNA) 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 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 GTP-bound form of another relasing factor, eRF3.

Domain 2 on eRF1 Recognizes the Stop CodonDomain 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 aminoacil-tRNA 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)

UGA C

UGA

RF2

Pausa

Frameshift(UGAC se lee como GAC y sigue +1)

GAC

RF2

Unión de RF2 terminación de la traducción

Experimento de DintzisDeterminación de la dirección de síntesis de las proteínas

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 theelectrophoretic 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?