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Antecedentes históricos

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

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Antecedentes históricos

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  1. 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

  2. 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

  3. Har Gobind Khorana

  4. 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 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

  5. “La molécula adaptadora”

  6. A partir de la secuencia de 300 tRNAs

  7. 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

  8. 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

  9. X174

  10. 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)

  11. Secuencia de reconocimiento de la iniciación de la traducción

  12. Small GTP-binding proteins require helper proteins, to • facilitate GDP/GTP exchange, or • promote GTP hydrolysis. Aguanine 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).

  13. AGTPase activating protein (GAP)causes a GTP-binding protein tohydrolyzeits bound GTP to GDP + Pi. Theactive sitefor 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

  14. 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-2is a small GTP-bindingprotein. IF-2-GTPbinds the initiator fMet-tRNAfMet & helps it to dock with the small ribosome subunit.

  15. 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. • 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 the large ribosomal subunitjoins the complex, GTP on IF-2 is hydrolyzed, leading to dissociation of IF-2-GDP and dissociation of IF-1. A domain of the largeribosomalsubunit serves as GAP (GTPase activating protein) for IF-2.

  16. 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.

  17. 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.

  18. EF-Tu-GTP binds & delivers an aminoacyl-tRNA to the A site on the ribosome. EF-Tu recognizes & bindsall 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

  19. 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.

  20. 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.

  21. 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

  22. 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.

  23. EF-Ts functions as GEF to reactivate EF-Tu. Interaction with EF-Ts causes EF-Tu to releaseGDP. Upon dissociation of EF-Ts, EF-TubindsGTP, which is present in the cytosol at higher concentration than GDP.

  24. 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.

  25. The 23S rRNA may be considered a "ribozyme."  As part of the reaction a proton (H+) is extracted from the attacking amino N.

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

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

  28. 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.

  29. 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.

  30. 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

  31. En eucariotes Iniciación

  32. 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

  33. Hipótesis Regulación de la actividad eIF-4E • eIF-4E es el IF en menor nivel (determinante) • Se regula a nivel de • Transcripción • Modificación (fosforilación ) • Inhibición

  34. Regulation of Step 1 • 1. Phosphorylation of the eIF4E BindingProteins, the 4E-BPs. • 2. Binding of PolyAdenylateBindingProtein (PABP) to eIF4G. Why? Because this circularizes the polysome, and allows ribosomal subunits to start new ribosomes.

  35. MAPK-Dependent Phosphorylation of eIF4E Is Mediated by the eIF4G Associated KinaseMnk 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

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