Traducción

El ribosoma es una ribozima!!

3 acidos nucleicos :mRNA, rRNA, tRNA

Tamaño relativo del ribosoma y los

tRNAs, y el mensajero
 ¿Qué es una ribozima?
Schematic (A) and ribbon (B) diagrams depicting the crystal
structure of the full-length hammerhead ribozyme. The
sequence and secondary structure in Figure A is color-coded
to match the structural features shown in Figure B. The
cleavage site nucleotide, C-17, is shown in green, and various
helical stems and loops are denoted using several other colors.
Tertiary hydrogen bonding contacts are denoted as thin black
lines, and tertiary stacking interactions as thin green lines. See
Figure 2 for a detailed schematic representation of the active
site.
Figure 2 A proposed mechanism for hammerhead ribozyme catalysis. The
nucleotide implicated as a general base in the self-cleavage reaction, G-12, is
shown in red. The nucleotide implicated in general acid catalysis, G-8, is shown
in dark blue. The substrate RNA is black, and water molecules that may
participate in the reaction, playing the roles of specific base and specific acid
catalysts, are shown in magenta and cyan. The scissile phosphate is depicted as
an (unobserved) pentacoordinated oxyphosphorane. G-12 can abstract a proton
from the 2'-OH of the cleavage-site ribose only if the endocyclic nitrogen N1
becomes deprotonated (as shown). This may happen via simple ionization, or
through a (rare and transient) tautomerization to the enolic form (as shown). As
the 2'-proton (in yellow) is abstracted by G-12, the bond between the 2'O and the
phosphorus atom forms, and that between the phosphorus and the 5'O begins to
break. As the latter breaks, a negative charge accumulates on the leaving group
5'O. A proton relay may then take place in which the 5'O acquires the 2'-proton
from G-8, which is simultaneously replaced with that from an adjacent water
molecule or hydronium ion (as shown).
Llevan a cabo la formacion del enlace peptidico, pero son en su mayor parte
Acidos nucleicos!!

Ribosomas y particulas ribonucleoproteina

Ribosomas rRNAs Proteinas

Bacteriana (70S) 23 S=2904 bases 31

66% RNA 5 S = 120 bases
2.5 MDa

50 y 30 S 16S= 1542 bases 21

-------------------------------------------------------------------------------
Mamiferos (80S) 28S = 4718 bases
60% RNA 5.8 S = 160 bases 49
4.2 MDa 5 S= 120 bases

18S =1874 bases 33

2.5 MD  2500 KDa (T7= 90, RNAP= 500)

Insulin  5.8 KDa

Polisomas: multiples ribosomas pueden traducir el mismo mRNA

polypeptide
direction of synthesis

ribosome
 Elongacion

•Bastante bien estudiado en procariontes

Factor Prokaryotes Eukaryotes

initiator tRNA fMet Met
large subunit 50S (23S and 5S rRNA) 60S (28S, 5S, and 5.8S rRNA)
(35 proteins) (49 proteins)
small subunit 30S (16S rRNA) 40S (18S rRNA)
(21 proteins ) (33 proteins)
ribosome 70S 80S
No tanta diferencia en tamaño entre bacteria y eucariontes
 Sintesis de polipeptidos

NH3- Met Ala Val Pro Ala Ser Asp -COOH

5’-m 7GpppNNNNNNNNNNACCAUGGCAGUUCCAGCUAGCGAUNNN-3’

codon (codes for 1 of 20 amino acids)

•Los mRNAs se leen en la direccion de 5’ a 3 primo

•La sintesis de proteinas es del extremo amino al carboxilo terminal

Como se lleva a cabo la reaccion?

 Los triples
Existen 4 bases (A, C, G, and U)

La historia de Gamow

• codigo de 1 nucleotido 41 = 4 amino acidos

•Codigo de 2 nucleotidos 42 = 16 amino acidos
• Codigo de 3 nucletotidos 43 = 64 amino acidos

•El codigo del triple es el mas pequeño para los 20 amino

acidos
Cual de los siguientes procesos no se lleva a cabo de 5 a 3
primo
a) Replicacion
b) Sintesis quimica de DNA
c) Transcripcion
d) Translacion
 El codigo de los tripletes

1961-Marshall Nirenberg and Johann Heinrich Matthaei

Phe Phe Phe Phe

5’-UUUUUUUUUUUUUUUUUUUUUUUUU-3’

Gobind Khorana

repeated Ser Leu Ser Leu

dinucleotide
5’-UCUCUCUCUCUCUCUCUCUCUCUCU-3’

•if the codon was 2 or 4 nt then the protein would be a homopolypeptide

Leu Leu Leu Leu

repeated Ser Ser Ser Ser
trinucleotide
Phe Phe Phe Phe
5’-UUCUUCUUCUUCUUCUUCUUCUUC-3’
 Heinrich Matthaei

Marshall Nirenberg

Incorporation of 14C––labeled valine into protein in Escherichia coli extracts. Endogenous

incorporation of radioactive amino acids into protein in E. coli extracts was high. However, amino
acid incorporation ceased after incubation for ~40 min with DNase I. I found that I could freeze E.
coli extracts and thaw them without loss of activity, so I incubated E. coli extracts in the absence
of radioactive amino acids for 40 min, divided the extracts into small aliquots and froze them for
use later in different experiments. Endogenous incorporation of radioactive amino acids was
greatly reduced in such extracts, and addition of mRNA preparations from ribosomes clearly
stimulated amino acid incorporation into protein [16, 34 and 49]. Reproduced from Ref. [16].
Poly(U) greatly stimulates the incorporation of radioactive phenylalanine into poly-
phenylalanine [8]. Addition of poly(A) completely inhibits the mRNA activity of
poly(U) by the formation of double-stranded and triple-stranded helices. By contrast,
addition of poly(C) has little effect on the mRNA activity of poly(U). This experiment,
done in 1961, was the first the anti-sense RNA experiment [8].
The specificity of randomly ordered polynucleotide templates in
stimulating amino acid incorporation into protein in Escherichia
coli extracts. Only the minimum kinds of bases necessary for
template activity are shown, so many amino acids that respond
to randomly ordered polynucleotides composed of two or more
kinds of bases are omitted. The base compositions of RNA
codons were derived from these experiments [49]. Reproduced
from Ref. [49].

The theoretical frequencies of RNA codons in randomly ordered poly(AC) preparations that contain
different proportions of A and C, compared with the observed frequencies of incorporation of
radioactively labeled amino acids into protein. The codon for histidine contains one A and two Cs,
and the codons for asparagine and glutamine contain two As and one C. These results showed that
the code is a triplet code [18 and 19]. Reproduced from Ref. [18].
 It took us about a year to synthesize the 64
trinucleotides and test each against 20
radioactive aminoacyl-tRNA preparations to
determine the nucleotide sequences of RNA
codons [30, 33, 35, 36, 37, 38, 39 and 40].
Gobind Khorana and his colleagues synthesized
the 64 trinucleotides chemically and also
determined nucleotide sequences of some RNA
codons [44]. The green AUG corresponds to
methionine and N-formyl-methionine tRNA, an
initiator of protein synthesis [45]. The red
codons specify the termination of protein
synthesis [46, 47 and 48].

The hydrophobic amino acids Phe, Leu, Ile, Met and Val correspond to chemically similar codons that
have U as the second base. By contrast, the hydrophilic amino acids Tyr, His, Gln, Asn, Lys, Asp and Glu
correspond to codons with A as the second base. In addition, amino acids with chemically similar side
chains, such as Asp and Glu, and Asn and Gln, have chemically similar codons. Clearly, the arrangement
of codons and amino acids is not random.
En bacteria, los ribosomas empiezan la traduccion del mRNA antes de que la RNA
Polimerasa haya terminado de transcribirlo. En eucariontes, los mRNA son sintetizados
Y procesado en el nucleo y transportados al citoplasma para la traduccion en
los ribosomas
 RNA de transferencia

Como es que un triplete especifica la identidad de un amino acido?

El papel de tRNA (RNA de transferencia)

El tRNA se enlaza a su correspondiente amino acido y se llama

amino acil tRNA y la adicion de los amino acidos al tRNA es llevada
a cabo por la aminoacyl tRNA sintetasa

Para cada uno de los 20 amino acidos hay una aminoacyl tRNA
sintetasa y uno o mas tRNAs

El 3’ del tRNA se genera al cortarse y
Adicionarse CCA
Transfer RNA is unique among nucleic acids in its
content of "unusual" bases. An unusual base is any
purine or pyrimidine ring except the usual A, G, C, and
U from which all RNAs are synthesized. All other bases
are produced by modification of one of the four bases
after it has been incorporated into the
polyribonucleotide chain
CCA Carga de los
amino acidos

Secuencia CCA
Anticodon; aparea con el mRNA (codon)

(anticodon)
Existen 61 different codons y
solamente 40 tRNAs.
La hipotesis de wobble
hypothesis : en la tercera posicion
se puede formar un par
no standar inosine se aparea con 3
bases
 The wobble position

Phe Leu

aminoacyl-tRNA

Phe Leu

•El mismo “ aminoacyl-tRNA” reconoce multiples codones

Wobble base pairs

codon anticodon
(third base) (first base)
AMINO ACIL tRNA sintetasas
 Dos sitios activos en la edicion y sintesis
de tRNAs

Class I (Glu-tRNA synthetase)/

Class II (Asp-tRNA synthetase)
Los ribososomas tienen 3
sitios de union al tRNA
A site :el aminoacyl-tRNA entrante se aparea
con el codon
P site: Es el sitio ocupado por el peptidyl-tRNA,
el tRNA que lleve el polipeptido nascente
E site: “Exit”

• Los ribosomas tienen 3 sitios:

• A site: “acceptor” …el
aminoacyl-tRNA se enlaza al
condon del mRNA
• P site: donde se ubica el tRNA
que elonga la cadena
polipetidica
• E site: donde el tRNA sale del
ribosoma
• Iniciación Empieza en el codon AUG

• En bacteria se tiene el sitio de union a ribosoma (secuencia Shine-Dalgarno) que

es complementaria a una secuencia del RNA ribosomal de la subunidad
pequeña
• Este sitio tiene la secuencia 5'-AGGAGGU-3' y se localiza ~6 nucleotidos rio
arriba del AUG inicial
Aminoacylated tRNAs occupy the P and A sites
LA INICIACION REQUERIE DE FACTORES

En bacteria se necesita de los 3

Factores IF-1, IF-2 y IF-3

Once the small ribosomal subunit is

bound to the mRNA, the aminoacyl
initiator tRNA binds to the AUG sequence.
The initiator methionine is modified with a
formyl group and is called N-formylmethionine.
 Initiation involves base pairing between mRNA and rRNA

Multiple genes on one mRNA initiate independently

The AUG is preceded by

a Shine-Dalgarno sequence
 Elongacion

Al principio de la elongacion, el tRNA

iniciador se encuentra en el sitio P, y los
sitios E y A se encuentran vacios. El
aminoacyl tRNA se enlaza al codon en el
sitio A y se lleva a cabo la formacion del
enlace peptidico
El factor de elongacion Tu carga a los aminoacyl-tRNA en el sitio A

Tu
Unido al GTP o al GDP
 EF-Tu directs binding of aminoacyl-tRNAs at the A site

ternary complex

gunanine nucleotide exchange

factor for EF-Tu

ribosome-dependent GTPase
activity of EF-Tu
 13 Elongation factors bind alternately to the ribosome

EF-G structure mimics

aminacyl-tRNA

EF factors have alternating interactions

Fidelidad elongacion
 Termination codons are recognized by protein factors

eRF1 mimics tRNA

Several factors have similar shapes
 eRF1 mimics a tRNA

Eukaryotes

Participates in the hydrolysis of the ester bond between the peptide and the tRNA
In the neighboring peptidyl-tRNA in the P site of the ribosome
 Requerimentos de energia de la traduccion

Step Event Energy

amino acid activation 1 ATP
Initiation …inactive 80S ribosome…dissociation -
mRNA binding to 40S subunit -
assembly of 40S and 60S subunits -
Elongation binding of aminoacyl-tRNA 1 GTP (EF-Tu)
peptide bond formation exergonic
translocation 1 GTP (EF-G)
Termination 1 GTP (RF-3)

1 ATP + 3 GTP / cycle

Estructura secundaria del RNA ribosomal

Yusupov et al. Science 292, 883 (2001)

El plegado en tres dimensiones del ribosoma 70S

Yusupov et al. Science 292, 883 (2001)

Cryo-EM map of the E. coli 70S ribosome

Gabashvili et al. Cell 100, 537 (2000)

X-ray structure of the 70S ribosome from T. thermophilus

Yusupov et al. Science 292, 883 (2001)

X-ray structure: tRNA binding sites

Yusupov et al. Science 292, 883 (2001)

 12 intersubunit contacts hold together the 70S ribosome

Magenta: RNA-RNA contacts

Yellow: protein-protein and protein-RNA contacts

Yusupov et al. Science 292, 883 (2001)

 The elongation process: the
peptidyl transferase center

deacetylated tRNA deacetylated tRNA analogue:

analogue: CCA
CCA
peptidyl tRNA analogue:
puromycin-Phe-caproic acid-
biotin

Schmeing et al. Nat. Struct. Biol. 9, 225-230 (2002)

tRNA-ribosome interactions

Yusupov et al. Science 292, 883 (2001)

Elongation: decoding of the mRNA

Ogle et al. Science 292, 897-902 (2001)

 Conservation in the 50S subunit

RNA: shown in space filling spheres Blue: conserved RNA in all species
Protein: shown as ribbons Green: non-conserved RNA
Moore and Steitz, Nature 418, 229-235 (2002)
One of the A-site
substrates, cytidine-cytidine-hydroxypuromycin (CChPmn),
includes C74; the other, ChPmn, does not.
 The mechanism by which the ribosome protects the peptidyltRNA
from hydrolysis can be understood by inspecting the conformation
of the peptidyl-tRNA analogue bound to the large ribosomal
subunit.

hidrólisis?
Como se evita la
Ciencia básica y antibioticos
Antibiotics that inhibit protein synthesis by binding to ribosomes
El sitio de union de los antibioticos es en el sitio activo

Steitz, T. A. and Moore, P. B. Trends Bio. Sci. 28, 411-418 (2003)

V
 New antibiotics for Serious Drug Resistant Infections

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•Experimentos para determinar la estructura del ribosoma

•Microscopia electronica

•Cristalizacion
A. Yonath, Weizman Institute of Science, Isreal
V. Ramakrishnan, MRC Lab of Molecular Biology, England
T. Steitz, Yale University, USA
Un poco de historia

Yonath
Steitz
The Steitz group utilized the
TaBr, W11Rh, and W18As
clusters to successfully phase
the structure of the large 50S
ribosomal subunit at low resolution