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TraduccinEl ribosoma es una ribozima!!

3 acidos nucleicos :mRNA, rRNA, tRNA

Tamao relativo del ribosoma y los

tRNAs, y el mensajero


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Qu es una ribozima?

Schematic (A) and ribbon (B) diagrams depicting the crystalstructure of the full-length hammerhead ribozyme. Thesequence and secondary structure in Figure A is color-codedto match the structural features shown in Figure B. Thecleavage site nucleotide, C-17, is shown in green, and varioushelical stems and loops are denoted using several other colors.Tertiary hydrogen bonding contacts are denoted as thin blacklines, and tertiary stacking interactions as thin green lines. SeeFigure 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, isshown in red. The nucleotide implicated in general acid catalysis, G-8, is shownin dark blue. The substrate RNA is black, and water molecules that mayparticipate in the reaction, playing the roles of specific base and specific acidcatalysts, are shown in magenta and cyan. The scissile phosphate is depicted asan (unobserved) pentacoordinated oxyphosphorane. G-12 can abstract a protonfrom the 2'-OH of the cleavage-site ribose only if the endocyclic nitrogen N1becomes deprotonated (as shown). This may happen via simple ionization, orthrough a (rare and transient) tautomerization to the enolic form (as shown). Asthe 2'-proton (in yellow) is abstracted by G-12, the bond between the 2'O and thephosphorus atom forms, and that between the phosphorus and the 5'O begins tobreak. As the latter breaks, a negative charge accumulates on the leaving group5'O. A proton relay may then take place in which the 5'O acquires the 2'-protonfrom G-8, which is simultaneously replaced with that from an adjacent watermolecule or hydronium ion (as shown).


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Ribosomas y particulas ribonucleoproteina

Ribosomas rRNAs Proteinas

Bacteriana (70S) 23 S=2904 bases 3166% RNA 5 S = 120 bases2.5 MDa

50 y 30 S 16S= 1542 bases 21-------------------------------------------------------------------------------Mamiferos (80S) 28S = 4718 bases60% RNA 5.8 S = 160 bases 494.2 MDa 5 S= 120 bases

18S =1874 bases 33

Llevan a cabo la formacion del enlace peptidico, pero son en su mayor parteAcidos nucleicos!!

2.5 MD 2500 KDa (T7= 90, RNAP= 500)

Insulin 5.8 KDa


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ElongacionBastante bien estudiado en procariontes

Factor Prokaryotes Eukaryotesinitiator tRNA fMet Metlarge 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


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No tanta diferencia en tamao entre bacteria y eucariontes


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Sintesis de polipeptidos

5-m7GpppNNNNNNNNNNACCAUGGCAGUUCCAGCUAGCGAUNNN-3Met Ala Val Pro Ala Ser AspNH3- -COOH

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

La sintesis de proteinas es del extremo amino al carboxilo terminal

codon (codes for 1 of 20 amino acids)

Como se lleva a cabo la reaccion?


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Los triplesExisten 4 bases (A, C, G, and U)La historia de Gamow codigo de 1 nucleotido 41 = 4 amino acidosCodigo de 2 nucleotidos 42 = 16 amino acidos Codigo de 3 nucletotidos 43 = 64 amino acidosEl codigo del triple es el mas pequeo para los 20 aminoacidosCual de los siguientes procesos no se lleva a cabo de 5 a 3primo

a) Replicacionb) Sintesis quimica de DNAc) Transcripciond) Translacion


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El codigo de los trip letes1961-Marshall Nirenberg and Johann Heinrich Matthaei

5-UUUUUUUUUUUUUUUUUUUUUUUUU-3Phe Phe Phe Phe

Gobind Khorana

5-UCUCUCUCUCUCUCUCUCUCUCUCU-3Ser Leu Ser Leuepeateddinucleotide

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

5-UUCUUCUUCUUCUUCUUCUUCUUC-3Phe Phe Phe Phe

repeatedtrinucleotide Ser Ser Ser SerLeu Leu Leu Leu


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Incorporation of 14Clabeled valine into protein in Escherichia coli extracts. Endogenousincorporation of radioactive amino acids into protein in E. coli extracts was high. However, aminoacid 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 absenceof radioactive amino acids for 40 min, divided the extracts into small aliquots and froze them foruse later in different experiments. Endogenous incorporation of radioactive amino acids was

greatly reduced in such extracts, and addition of mRNA preparations from ribosomes clearlystimulated amino acid incorporation into protein [16, 34 and 49]. Reproduced from Ref. [16].

Heinrich MatthaeiHeinrich Matthaei

Marshall Nirenberg


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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].
http://lib3.tufts.edu:2061/science?_ob=ArticleURL&_udi=B6TCV-4B84YVY-1&_coverDate=12%2F18%2F2003&_rdoc=1&_fmt=full&_orig=search&_sort=d&view=c&_acct=C000014058&_version=1&_urlVersion=0&_userid=201547&md5=5968db711645e2daf770de1b19a044b8http://lib3.tufts.edu:2061/science?_ob=ArticleURL&_udi=B6TCV-4B84YVY-1&_coverDate=12%2F18%2F2003&_rdoc=1&_fmt=full&_orig=search&_sort=d&view=c&_acct=C000014058&_version=1&_urlVersion=0&_userid=201547&md5=5968db711645e2daf770de1b19a044b8http://lib3.tufts.edu:2061/science?_ob=ArticleURL&_udi=B6TCV-4B84YVY-1&_coverDate=12%2F18%2F2003&_rdoc=1&_fmt=full&_orig=search&_sort=d&view=c&_acct=C000014058&_version=1&_urlVersion=0&_userid=201547&md5=5968db711645e2daf770de1b19a044b8http://lib3.tufts.edu:2061/science?_ob=ArticleURL&_udi=B6TCV-4B84YVY-1&_coverDate=12%2F18%2F2003&_rdoc=1&_fmt=full&_orig=search&_sort=d&view=c&_acct=C000014058&_version=1&_urlVersion=0&_userid=201547&md5=5968db711645e2daf770de1b19a044b8


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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]. Reproducedfrom 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].


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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.

It took us about a year to synthesize the 64

trinucleotides and test each against 20

radioactive aminoacyl-tRNA preparations todetermine 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].


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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 llamaamino acil tRNA y la adicion de los amino acidos al tRNA es llevadaa cabo por la aminoacyl tRNA sintetasa

Para cada uno de los 20 amino acidos hay una aminoacyl tRNA

sintetasa y uno o mas tRNAs


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Transfer RNA is unique among nucleic acids in itscontent of "unusual" bases. An unusual base is anypurine or pyrimidine ring except the usual A, G, C, andU from which all RNAs are synthesized. All other basesare produced by modification of one of the four basesafter it has been incorporated into the

polyribonucleotide chain

El 3 del tRNA se genera al cortarse y

Adicionarse CCA
http://www.ergito.com/441http://www.ergito.com/441


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(anticodon)

CCA Carga de los

amino acidos

Secuencia CCAAnticodon; aparea con el mRNA (codon)


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Existen 61 different codons ysolamente 40 tRNAs.

La hipotesis de wobblehypothesis : en la tercera posicionse puede formar un par

no standar inosine se aparea con 3bases


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The wobble position

Phe

Phe Leu

Leu

aminoacyl-tRNA

El mismo aminoacyl-tRNA reconoce multiples codones


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Wobble base pairsanticodon(first base)codon(third base)


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AMINO ACIL tRNA sintetasas


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Dos sitios activos en la edicion y sintesisde tRNAs

Class I (Glu-tRNA synthetase)/

Class II (Asp-tRNA synthetase)


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Los ribososomas tienen 3


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Los ribososomas tienen 3sitios de union al tRNA


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A site :el aminoacyl-tRNA entrante se apareacon el codonP site: Es el sitio ocupado por el peptidyl-tRNA,

el tRNA que lleve el polipeptido nascenteE site: Exit

Los ribosomas tienen 3 sitios:

A site: acceptor el

aminoacyl-tRNA se enlaza alcondon del mRNA

P site: donde se ubica el tRNAque elonga la cadena

polipetidica E site: donde el tRNA sale del

ribosoma


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Iniciacin Empieza en el codon AUG


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p

En bacteria se tiene el sitio de union a ribosoma (secuencia Shine-Dalgarno) quees complementaria a una secuencia del RNA ribosomal de la subunidadpequea

Este sitio tiene la secuencia 5'-AGGAGGU-3' y se localiza ~6 nucleotidos rioarriba del AUG inicial


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Aminoacylated tRNAs occupy the P and A sites


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En bacteria se necesita de los 3Factores IF-1, IF-2 y IF-3

Once the small ribosomal subunit isbound to the mRNA, the aminoacylinitiator tRNA binds to the AUG sequence.The initiator methionine is modified with aformyl group and is called N-formylmethionine.

LA INICIACION REQUERIE DE FACTORES


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Elongacion

Al principio de la elongacion, el tRNA

iniciador se encuentra en el sitio P, y lossitios E y A se encuentran vacios. El

aminoacyl tRNA se enlaza al codon en el

sitio A y se lleva a cabo la formacion delenlace peptidico


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El factor de elongacion Tu carga a los aminoacyl-tRNA en el sitio A

Tu

Unido al GTP o al GDP


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EF-Tu directs binding of aminoacyl-tRNAs at the A site

gunanine nucleotide exchangefactor for EF-Tu

ternary complex

ribosome-dependent GTPaseactivity of EF-Tu


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13 Elongation factors bind alternately to the ribosome

EF-G structure mimicsaminacyl-tRNA

EF factors have alternating interactions

Fid lid d l i


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Fidelidad elongacion


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Termination codons are recognized by protein factors


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Several factors have similar shapeseRF1 mimics tRNA


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EukaryoteseRF1 mimics a tRNA

Partic ipates in the hydrolysis of the ester bond between the peptide and the tRNAIn the neighbor ing peptidyl-tRNA in the P site of the ribosome


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Requerimentos de energia de la traduccion

Step Event Energyamino acid activation 1 ATP

Initiation inactive 80S ribosomedissociation -mRNA binding to 40S subunit -assembly of 40S and 60S subunits -

Elongation binding of aminoacyl-tRNA 1 GTP (EF-Tu)peptide bond formation exergonictranslocation 1 GTP (EF-G)

Termination 1 GTP (RF-3)1 ATP + 3 GTP / cycle


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Yusupov et al. Science292, 883 (2001)

Estructura secundaria del RNA ribosomal


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Cryo-EM map of the E. co l i 70S ribosome

Gabashvili et al. Cell100, 537 (2000)

X-ray structure of the 70S ribosome from T. therm op hilu s


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X-ray structure: tRNA binding sites



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12 intersubunit contacts hold together the 70S ribosome


Magenta: RNA-RNA contactsYellow: protein-protein and protein-RNA contacts


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The elongation process: thepeptidyl transferase center

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

deacetylated tRNA analogue:CCAdeacetylated tRNAanalogue:CCApeptidyl tRNA analogue:puromycin-Phe-caproic acid-biotin


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tRNA-ribosome interactions



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Elongation: decoding of the mRNA

Ogle et al. Science292, 897-902 (2001)

Conservation in the 50S subunit


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RNA: shown in space f il ling spheres Blue: conserved RNA in al l speciesProtein: shown as ribbons Green: non-conserved RNAMoore and Steitz, Nature418, 229-235 (2002)


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One of the A-sitesubstrates, cytidine-cytidine-hydroxypuromycin (CChPmn),

includes C74; the other, ChPmn, does not.


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Themechanismb

yw

hichtheribosome

protectsthepeptidyltRNA

fromh

ydrolysiscanb

eunderstoodbyinspectingthecon

formation

ofthepeptidyl-tRNA

analogueboundtothelargeribosom

al

subunit.

Como se evita lahidrlisis?


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Cienciabsica

yantibioticos


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vo


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Elstoduodoabocoeeestoa

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


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V


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New antibiotics for Serious Drug Resistant Infections


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New antibiotics for Serious Drug Resistant Infections

Recent Rib-X News09/13/10

Rib-X Pharmaceuticals Presents Data SupportingDelafloxacin as a Potential Best-in-class Fluoroquinoloneat ICAAC

09/13/10

Rib-X Pharmaceuticals' Nobel Prize Winning Structure-based Design and Optimization Platform to be Featuredin a Symposium Reviewing 50 Years of Antibiotic DrugDiscovery at the 50th ICAAC Meeting

About UsRib-X Pharmaceuticals, Inc., is a product-driven small-molecule drug discovery and development company focusedon developing and commercializing antibiotics to treat highly-

resistant bacterial infections.Read MoreQuick Link to Pubs and Posters

PipelineDelafloxacinRadezolidR-02R-04

Experimentos para determinar la estructura del ribosomaMicroscopia electronicaCristalizacion

A. Yonath, Weizman Institute of Science, Isreal

V. Ramakrishnan, MRC Lab of Molecular Biology, EnglandT. Steitz, Yale University, USA

U d hi i
http://www.rib-x.com/news_and_events/press_releaseshttp://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/about_ushttp://www.rib-x.com/about_ushttp://www.rib-x.com/r_and_d/publications_and_presentationshttp://www.rib-x.com/pipelinehttp://www.rib-x.com/pipeline/rx_3341http://www.rib-x.com/pipeline/rx_1741http://www.rib-x.com/pipeline/rx_02http://www.rib-x.com/pipeline/rx_02http://www.rib-x.com/pipeline/rx_02http://www.rib-x.com/pipeline/rx_04http://www.rib-x.com/pipeline/rx_04http://www.rib-x.com/pipeline/rx_04http://www.rib-x.com/pipeline/rx_04http://www.rib-x.com/pipeline/rx_04http://www.rib-x.com/pipeline/rx_04http://www.rib-x.com/pipeline/rx_04http://www.rib-x.com/pipeline/rx_02http://www.rib-x.com/pipeline/rx_02http://www.rib-x.com/pipeline/rx_02http://www.rib-x.com/pipeline/rx_02http://www.rib-x.com/pipeline/rx_1741http://www.rib-x.com/pipeline/rx_3341http://www.rib-x.com/pipelinehttp://www.rib-x.com/r_and_d/publications_and_presentationshttp://www.rib-x.com/about_ushttp://www.rib-x.com/about_ushttp://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/news_and_events/release_2010_09_13http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/release_2010_09_14http://www.rib-x.com/news_and_events/press_releaseshttp://www.rib-x.com/news_and_events/press_releaseshttp://www.rib-x.com/news_and_events/press_releaseshttp://www.rib-x.com/news_and_events/press_releases


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Un poco de historia

Yonath


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Steitz


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The Steitz group utilized the

TaBr, W11Rh, and W18Asclusters to successfully phasethe structure of the large 50Sribosomal subunit at low resolution


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