TraducciónIII. Elongación, Terminación, Modificaciones de proteínas

1. Leer en el libro GENES IX la parte correspondiente a elongación y terminación de la traducción: p. 167-185 GENES IX.

2. Entender la secuencialidad de cada paso de la Elongación y el rol de cada una de los componentes involucrados (factores de elongación; subunidades ribosomales; aa-tRNAs; mRNA). La elongación es muy similar entre procariontes y eucariontes.

3. Saber qué eventos implica la terminación y qué modificaciones de proteínas ocurren durante la traducción.

4. Ver el video de Traducción subido en las páginas. Ser capaces de calcular la energía total que gasta el proceso de Traducción de una proteína.

5. Contestar el cuestionario correspondiente a la Clase IV que estará disponible en el Classroom.

El ribosoma competente para elongar presenta:

Las dos subunidades unidas

mRNA colocado entre las subunidades

El sitio P ocupado por tRNA-Met

El ribosoma al terminar el inicio

Se requiere para la Elongación

• Contar con una reserva de aa-tRNAs elongadores cargadoscorrectamente.

• Muchos Ribosomas• Mucha energía en forma de GTP• Factores de elongación (proteínas abundantes)

El papel de los factores de elongaciónBacteria Eucariontes

EF-Tu

EF-Ts

EF-G

eEF-1A

eEF-1B

eEF-2

Llevar el aa-tRNA al sitio A; GTP (TC)

GEF para EF-Tu/eEF-1A (GDPàGTP)

Translocación; GTP

Pasos de la Elongación

1. Entrada de un aa-tRNA correcto (correspondiente al codón) en el sitio A del ribosoma. GASTO de 1 GTP2. Formación del enlace peptídico.3. Translocación del ribosoma exactamente 3 bases (1 codón) en el sentido 5’ à 3’. GASTO de 1 GTP

Estos pasos se repiten tantas veces como aminoácidos haya que agregar.

1. Entrada de aa-tRNA al sito A

a. El aa-tRNA es llevado al Ribosoma en forma de complejo ternario (TC) EFTu-GTP-aa-tRNA

b. El centro decodificador (30S) revisa apareamientoc. Se hidroliza GTP y sale EFTu-GDPd. El aa-tRNA se acomoda

Recuperación de EFTu-GTP

EF-Tu-GTP

EF-Tu-GDP + Pi

EF-Tu · EF-Tu + GDP

EF-Tu-GTP + EF-Ts

· EF-Ts

· GTP

2. Formación del enlace peptídico

Es un paso muy rápidoLa cadena peptídica pasa al tRNA de sitio AEl tRNA de sitio P queda vacío

Formación del enlace peptídico (Ribozima)

El entorno del rRNA 23S/28S, después del acomodamiento, posibilitael ataque nucleofílico del grupo amino del aa-tRNA del sitio A al carbono del grupo carboniloesterificado al tRNA sitio P.

NH2

3. Translocación

Zhang et al. Science 2009

Subunidades 30S y 50S

a. Hay un movimiento de la subunidad 30S que propicia el estado E/P; P/A de PRE-translocación.

3. Translocaciónb. Se completa la translocación cuando entra EF-G y se hidroliza una molécula de GTP.

Terminación de la Traducción

- Reconocer el STOP codon

- Liberar la proteínasintetizada

- Desensamblar el ribosoma, el mRNA y tRNA del sitio P

GASTO de 1 GTP

Factores de terminación

Bacteria Eucariontes

RF1RF2RF3

RRF

eRF1

eRF3

Rli levaduraABCE humano

• RF1, RF2 ó eRF1: reconocen codones STOP; promueven hidrólisis del enlace entre la proteína recién sintetizada y el último tRNA

• RF3 ó eRF3: GTPasa que ayuda a liberar a RF1/RF2 ó eRF1

• RRF ó Rli/ABCE: desensamblado y reciclado de subunidades ribosomales

Mímicas moleculares dentro del ribosoma

- El ribosoma es una máquina molecular que da cabida escencialmente al mRNA y a moléculas de aa-tRNA. Otros componentes como los factores de traducción que deben entrar al ribosoma, asemejan la estructura de un tRNA aminoacilado.

- Incluso hay combinaciones de aminoácidos en la estructura proteica de algunos factores (RF2, eRF1) que simulan una interacción con el codón (rojo) o con el entorno de la región aminoacilada (verde) del tRNA.

Günter et al., 2009; Nat Struct & Mol Biol 16, 589 - 597

Plegamiento de proteínas¿Cómo interviene el ribosoma en el plegamiento co-traduccional de proteínas?

• Tiene el canal de salida del péptido naciente (poco o nulo plegamiento)

• Une a proteínas que interaccionan con el amino terminal de la proteína que va saliendo

a) Remoción del grupo formilo de la metionina N-terminal de la bacteria; eliminación de la metionina

b) Eliminación de la metionina N-terminal en algunas proteínas eucariontes; acetilación N-terminal

Modificaciones co-traduccionales del extremo N-terminal

Plegamiento asistido por chaperonas en bacteria

a) El factor Trigger (TF) se une al péptido naciente y al ribosoma para actuar como chaperona de plegamiento; algunas proteínas requieren la ayuda de otros sistemas de chaperonas como Hsp70 (DnaJ/DnaK) o Hsp60 (GroEL/GroES)

b) TF se asocia directamente a proteínas ribosomales (pr) que rodean la salida del péptido naciente; L17 y L32 son pr específicas de bacteria; PDF (deformilasa)

¿Es posible el plegamiento correcto de proteínas sin las chaperonas?

Direccionamiento co-traduccional de proteínas destinadas a retículo endoplásmico (RE)

Cuando el péptido señal de la proteína que debe ser direccionada emerge del ribosoma, la traducción se detiene para llevar el péptido naciente a la membrana del RE y llevarlo al lumen. Arranca de nuevo la traducción y la proteína permanece siempre en el lumen. El péptido señal se corta por una peptidasa de RE de manera co-traduccional.

Reticulo Endoplásmico Rugoso

Péptido señal

Peptidasa

Glicosilación co-traduccional de proteínas en RE

¿Cuál es la función de la glicosilación en las proteínas que la llevan?

Secuencias señal determinan el destino de las proteínas

KDEL

- Epéptido señal para RE está en el extremo NH2, se reconoce co-traduccionalmente y se corta

- Para mitochondria y cloroplastos el péptido señal está en el extremo NH2, se reconocepost-traduccionalmente y se corta

- Para el núcleo las señales, está en cualquier parte de la proteína y NO se eliminan

Pausado de los ribosomas en Elongación

et al., 2010). Translation of multiple synonymous codons by asingle tRNA has been demonstrated to occur by wobble base-pairing: standard Watson-Crick base-pairing (A-U, G-C) isrequired at the first and second positions of a codon, and‘‘wobbling’’ (e.g., G-U) is allowed at the third position of a codon(corresponding to the 50 position of the anticodon, i.e., position34 of a tRNA) (Crick, 1966; Soll et al., 1966). However, the affinityby which synonymous codons are recognized via wobble base-pairing is not similar. For instance, tRNAs with G in the 50 positionof the anticodon have a higher binding affinity for C-endingcodons than for U-ending codons (Crick, 1966; Soll et al., 1966).

The influence of wobble base-pairing on decoding rates ofcodons by the ribosome is still unresolved and complex toanalyze. The translation kinetics of different codon-anticodonpairs are complex, as the following processes can play a role:(1) the diffusion kinetics of the matching tRNA, (2) the relativecodon-binding affinity of matching tRNAs over mismatchingtRNAs (Gromadski et al., 2006), and (3) the translocation kineticsof mRNA and tRNA through the ribosome, which are affected byanticodon-codon interactions (Khade and Joseph, 2011).Recently conflicting results were published that reported eitherslower (Stadler and Fire, 2011) or faster (Gardin et al., 2014) de-coding of wobbling codons. This process deserves a moredetailed analysis of both data sets and methods and shouldalso take into account the effect of tRNA modifications onwobble base-pairing.

Modified nucleotides present in tRNAs further extend therange of recognized synonymous codons by affecting the abilityof these tRNAs to form wobble base pairs (Agris et al., 2007).Some specific, key tRNA modifications are present in onlysome domains of life (Grosjean et al., 2010). First, a key tRNAmodification, present in eukaryotes and to some extent in bacte-ria, is the modification of adenine-34 to inosine-34, which allowsnon-Watson-Crick pairing with adenine, cytosine, and uridine.Second, exclusively in bacteria, the key tRNA modifications of

uridine-34 to hydroxy-uridine and derivatives are found, allowingwobble pairing with adenine, guanosine, and uridine. These keytRNA modifications explain many differences in the tRNA setsthat are present in archaea, bacteria, and eukaryotes (Table 1)(Novoa et al., 2012).Correlation of Codon Bias with tRNA PoolsAfter the discovery of codon bias, a positive correlation wasfound between the frequency of codons and the concentrationof tRNAs with complementary anticodons (Figure 2A) (Ikemura,1985). This fact was established for several prokaryotes andunicellular eukaryotes (Kanaya et al., 1999). However, this corre-lation initially could not be identified for several, mostly multicel-lular eukaryotes.To better analyze the relation between codon frequency bias

and tRNA abundance in multicellular organisms, the tRNA adap-tation index (tAI) was developed (dos Reis et al., 2004). Thismetric is based on the copy number of tRNA genes, assumedto be correlated with tRNA abundance in cells, and also takesinto account the efficiency of codon-anticodon binding, relatedto Crick’s wobble rules (Crick, 1966). On the basis of computa-tional analyses, it was concluded that organisms with largergenomes have higher tRNA gene redundancy, which woulddecrease selection for specific codons (dos Reis et al., 2004).This explained why in multicellular organisms with larger ge-nomes, no positive correlation between codon usage andtRNA abundance could be identified in many studies. However,most studies at that time estimated tRNA abundance on thebasis of tRNA gene copy numbers.Correlations based on tRNA copy numbers do not take into

account that pools of distinct tRNAs and aminoacyl-tRNA spe-cies are dynamic and can vary considerably in different condi-tions. For example, it was demonstrated by microarray analysisthat tRNA expression abundance in humans varies widelyamong different tissues. This abundance could be statisticallycorrelated to codon usage of highly expressed genes specificfor those tissues (Dittmar et al., 2006). Furthermore, in bacteria,it was found that the charging levels of different tRNAs recog-nizing synonymous codons vary drastically in response to aminoacid starvation; that is, although the pool of some synonymoustRNAs remains completely charged, the charged fraction ofothers can decline to zero (Dittmar et al., 2005; Elf et al., 2003).So far, codon frequencies were correlatedmostly with the total

supply of tRNAs. However, when a more frequently used codonis recognized by a more abundant tRNA species, this codon willalso compete for this tRNA with more codons. To take this intoaccount, the normalized translation efficiency (nTE) metric wasintroduced, correcting for supply as well as demand rates oftRNAs (Pechmann and Frydman, 2013). The nTE considerscodons to be more optimal if their relative tRNA abundance onthe basis of gene copy number (supply) exceeds their relativecognate codon usage (demand) on the basis of codon fre-quencies in mRNA. Although the tAI and nTE already givegood indications of the availability of tRNAs for the translationof synonymous codons, this approximation could still beimproved. The actual important value is the level of mature ami-noacyl-tRNAs ready for amino acid delivery in the translationprocess. However, because the levels of tRNA expression andcharging can undergo major fluctuations on the basis of cellular

Figure 1. Translation in the Ribosome and tRNA StructureCartoon of the ribosome (green) during translation of a mRNA (blue) with awobbling codon-anticodon base pair encoding a leucine amino acid. A site,aminoacyl-tRNA site; E site, exit site; P site, peptidyl-tRNA site.

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condition, it is not straightforward to take these values into ac-count. In addition, it was demonstrated that codon frequencybias can be better correlated with tRNA gene frequencies in alldomains of life, if two major, domain-specific tRNA modificationtypes are taken into account (Novoa et al., 2012).

To summarize, highly expressed proteins are generally en-coded by genes that contain relatively high proportions of co-dons recognized by abundant, charged tRNAs with kineticallyefficient codon-anticodon base-pairing. This explains to a largeextent the observed codon frequency bias in many genes andgenomes. In addition to codon frequency bias, two other generaltypes of codon bias have been identified in recent years: codonpair bias and codon co-occurrence bias; these will be discussedhereafter.Synonymous Codon Co-occurrence BiasRecently it was shown that not only the overall frequency of syn-onymous codons but also the order in which they reside in a geneare biased. While studying all coding sequences of Saccharo-myces cerevisiae, a bias was revealed of clustered synonymouscodons, called codon co-occurrence bias. Instead of a randomdistribution of synonymous codons on a coding sequence, thereis a bias to cluster those synonymous codons that are recog-nized by the same tRNA (i.e., identical codons and isoacceptingcodons) (Cannarozzi et al., 2010) (Figure 2B). The effect of co-occurrence bias involves both frequent and rare codons and ismost prominent in highly expressed genes that must be rapidlyinduced, such as those involved in stress response (Cannarozziet al., 2010).

It has been suggested that tRNAs remain in proximity to thetranslating ribosome after their exit from the E site and thatthey are subsequently recharged by the corresponding amino-acyl-tRNA synthetases that somehow co-localize with the ribo-

some (Cannarozzi et al., 2010; Godinic-Mikulcic et al., 2014).At the next occurrence of the same or isoaccepting codon, thecharged tRNA would be readily available for translation; thiswould have a positive effect on translation efficiency (Cannarozziet al., 2010).Co-occurrence bias has been demonstrated in eukaryotes,

bacteria, and archaea (Cannarozzi et al., 2010; Shao et al.,2012; Zhang et al., 2013). However, co-occurrence of identicalcodons is strongly biased in all domains of life, whereas co-occurrence of non-identical isoaccepting codons is less promi-nent in prokaryotes than in eukaryotes (Shao et al., 2012; Zhanget al., 2013). The fact that co-occurrence of non-identical isoac-cepting occursmore in eukaryotesmost likely correlates with dif-ferences in affinity of codon-anticodon pairs between eukaryotesand prokaryotes (Shao et al., 2012). Domain-specific key modifi-cations of tRNA result in differences in affinities of wobble base-pairing for certain synonymous codons. It has been hypothesizedthat only non-identical codon pairs that are recognized by a tRNAwith similarly high affinity may result in co-occurrence bias (Shaoet al., 2012). The described findings demonstrate that the use ofidentical and some isoaccepting codons in close proximity aregenerally advantageous for the translation process.Non-synonymous Codon Pair BiasIn addition to codon frequency and co-occurrence, also thecontext inwhichacodon resides is under selectiveconstraint.Nu-cleotides neighboring a particular codon are distributed in a non-randommanner (Buchanet al., 2006;GutmanandHatfield, 1989).This phenomenon is called codon pair bias (Figure 2C). Forexample, there are eight possible codon pairs to encode the adja-cent amino acids alanine and glutamate. On the basis of codonfrequencies, one would expect these amino acids to be encodedequally byGCC-GAAandGCA-GAGcodonpairs.However, in hu-mans, the GCC-GAA pair is heavily underrepresented comparedwith the expected frequency, even though it contains GCC, themost prevalent codon for alanine (Coleman et al., 2008). Somecodon pairs are universally avoided or preferred; for example,nnUAnn codon pairs are usually underrepresented, whereasnnGCnn codon pairs are most preferred (Tats et al., 2008).Although the exactmechanismbywhich codon pair biasmight

enhance translation efficiency is currently not well understood, itis assumed that tRNAs in the A and P sites of the ribosome caninteract and as such influence the efficiency of the translationprocess (Figure 2C) (Buchan et al., 2006). Several viral genomesalso contain codon pair bias, which generally matches that oftheir host. Modification of this codon pair usage in virulence-related genes of viruses and has been presented as an elegantstrategy to produce vaccines with attenuated viruses (Colemanet al., 2008). However, it was recently suggested that this atten-uation may be caused by an increased CpG and UpA dinucleo-tide bias rather than by a changed codon pair bias, becausethese dinucleotides are generally used at a low frequency inRNA and small DNA viruses infecting mammals and plants (Tul-loch et al., 2014).

Is Translation Efficiency Correlated with Codon Bias?The efficiency of translation, and the resulting protein produc-tion, is determined by both translation initiation and elongationrates. While the translation initiation rate controls how often a

A

B C

Figure 2. Different Types of Codon Bias(A) Frequency bias will result in effective protein production when the fre-quency of used codons matches the cellular tRNA population.(B) Co-occurrence bias enhances protein expression, presumably because oftRNA recharging in the vicinity of the translating ribosome.(C) Pair bias is probably selected because of more optimal interactions oftRNAs in the A site and the P site.

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Teorías sobre el pausado de ribosomasen elongación

n Frecuencia del uso de codones (codon bias); presencia de codones raros

n Estructura secundaria en el mRNAn Presencia de secuencias Shine-Dalgarno like

¿Para qué se necesita bajar la velocidad de elongacióndel ribosoma?

• Plegamiento co-traduccional• Reconocer péptido señal• Translocación de la proteína a membrana