15.5: Ribosomas y síntesis de proteínas

Habilidades para Desarrollar

• Describir los diferentes pasos en la síntesis de proteínas
• Discutir el papel de los ribosomas en la síntesis de proteínas

La síntesis de proteínas consume más energía de una célula que cualquier otro proceso metabólico. A su vez, las proteínas representan más masa que cualquier otro componente de los organismos vivos (con la excepción del agua), y las proteínas realizan prácticamente todas las funciones de una célula. El proceso de traducción, o síntesis de proteínas, implica la decodificación de un mensaje de ARNm en un producto polipeptídico. Los aminoácidos se encadenan covalentemente mediante enlaces peptídicos entrelazados en longitudes que van desde aproximadamente 50 residuos de aminoácidos hasta más de 1,000. Cada aminoácido individual tiene un grupo amino (NH ₂) y un grupo carboxilo (COOH). Los polipéptidos se forman cuando el grupo amino de un aminoácido forma un enlace amida (es decir, péptido) con el grupo carboxilo de otro aminoácido (Figura\(\PageIndex{1}\)). Esta reacción es catalizada por los ribosomas y genera una molécula de agua.

Figura\(\PageIndex{1}\): Un enlace peptídico une el extremo carboxilo de un aminoácido con el extremo amino de otro, expulsando una molécula de agua. Por simplicidad en esta imagen, solo se muestran los grupos funcionales involucrados en el enlace peptídico. Las designaciones R y R' se refieren al resto de cada estructura de aminoácidos.

La maquinaria de síntesis de proteínas

Además del molde de ARNm, muchas moléculas y macromoléculas contribuyen al proceso de traducción. La composición de cada componente puede variar entre especies; por ejemplo, los ribosomas pueden consistir en diferentes números de ARNr y polipéptidos dependiendo del organismo. Sin embargo, las estructuras y funciones generales de la maquinaria de síntesis de proteínas son comparables de bacterias a células humanas. La traducción requiere la entrada de un molde de ARNm, ribosomas, ARNt y varios factores enzimáticos.

Enlace al aprendizaje

Haga clic en los pasos de este interactivo de PBS para ver la síntesis de proteínas en acción.

Ribosomas

Incluso antes de que se traduzca un ARNm, una célula debe invertir energía para construir cada uno de sus ribosomas. En E. coli, hay entre 10,000 y 70,000 ribosomas presentes en cada célula en un momento dado. Un ribosoma es una macromolécula compleja compuesta por ARNr estructurales y catalíticos, y muchos polipéptidos distintos. En eucariotas, el nucleolo está completamente especializado para la síntesis y ensamblaje de ARNr.

Los ribosomas existen en el citoplasma en procariotas y en el citoplasma y retículo endoplásmico rugoso en eucariotas. Las mitocondrias y los cloroplastos también tienen sus propios ribosomas en la matriz y el estroma, que se parecen más a los ribosomas procariotas (y tienen sensibilidades similares a los medicamentos) que los ribosomas justo fuera de sus membranas externas en el citoplasma. Los ribosomas se disocian en subunidades grandes y pequeñas cuando no están sintetizando proteínas y se reasocian durante el inicio de la traducción. En E. coli, la subunidad pequeña se describe como 30S, y la subunidad grande es 50S, para un total de 70S (recuerde que las unidades de Svedberg no son aditivas). Los ribosomas de mamíferos tienen una subunidad 40S pequeña y una subunidad 60S grande, para un total de 80. La subunidad pequeña es responsable de unirse al molde de ARNm, mientras que la subunidad grande se une secuencialmente a ARNt. Cada molécula de ARNm es traducida simultáneamente por muchos ribosomas, todos sintetizando proteínas en la misma dirección: leyendo el ARNm de 5' a 3' y sintetizando el polipéptido desde el extremo N hasta el extremo C. La estructura completa de ARNM/poli-ribosoma se denomina polisoma.

ARNt

Los ARNt son moléculas de ARN estructural que fueron transcritas a partir de genes por la ARN polimerasa III. Dependiendo de la especie, existen de 40 a 60 tipos de ARNt en el citoplasma. Sirviendo como adaptadores, los ARNt específicos se unen a secuencias en el molde de ARNm y agregan el aminoácido correspondiente a la cadena polipeptídica. Por lo tanto, los ARNt son las moléculas que realmente “traducen” el lenguaje del ARN al lenguaje de las proteínas.

De los 64 posibles codones de ARNm, o combinaciones de tripletes de A, U, G y C, tres especifican la terminación de la síntesis de proteínas y 61 especifican la adición de aminoácidos a la cadena polipeptídica. De estos 61, un codón (AUG) también codifica el inicio de la traducción. Cada anticodón de ARNt puede emparejarse con uno de los codones de ARNm y agregar un aminoácido o terminar la traducción, de acuerdo con el código genético. Por ejemplo, si la secuencia CUA ocurriera sobre un molde de ARNm en el marco de lectura adecuado, se uniría a un ARNt que expresara la secuencia complementaria, GAU, que estaría unida al aminoácido leucina.

Como moléculas adaptadoras de traducción, es sorprendente que los ARNt puedan encajar tanta especificidad en un paquete tan pequeño. Considerar que los ARNt necesitan interactuar con tres factores: 1) deben ser reconocidos por la aminoacil sintetasa correcta (ver más adelante); 2) deben ser reconocidos por los ribosomas; y 3) deben unirse a la secuencia correcta en el ARNm.

Aminoacil ARNt sintetasas

El proceso de síntesis de pre-ARNt por la ARN polimerasa III solo crea la porción de ARN de la molécula adaptadora. El aminoácido correspondiente debe agregarse posteriormente, una vez que el ARNt sea procesado y exportado al citoplasma. A través del proceso de “carga” de ARNt, cada molécula de ARNt se une a su aminoácido correcto por un grupo de enzimas llamadas aminoacil ARNt sintetasas. Al menos un tipo de aminoacil ARNt sintetasa existe para cada uno de los 20 aminoácidos; el número exacto de aminoacil ARNt sintetasas varía según la especie. Estas enzimas primero se unen e hidrolizan ATP para catalizar un enlace de alta energía entre un aminoácido y monofosfato de adenosina (AMP); una molécula de pirofosfato es expulsada en esta reacción. El aminoácido activado se transfiere luego al ARNt y se libera AMP.

El Mecanismo de Síntesis de Proteínas

Al igual que con la síntesis de ARNm, la síntesis de proteínas se puede dividir en tres fases: iniciación, elongación y terminación. El proceso de traducción es similar en procariotas y eucariotas. Aquí exploraremos cómo se produce la traducción en E. coli, un procariota representativo, y especificaremos cualquier diferencia entre la traducción procariota y eucariota.

Iniciación de la Traducción

La síntesis de proteínas comienza con la formación de un complejo de iniciación. En E. coli, este complejo involucra el pequeño ribosoma 30S, el molde de ARNm, tres factores de iniciación (IF; IF-1, IF-2 e IF-3) y un ARNt iniciador especial, llamado\(\text{tRNA}_\text{f}^\text{Met}\). The initiator tRNA interacts with the start codon AUG (or rarely, GUG), links to a formylated methionine called fMet, and can also bind IF-2. Formylated methionine is inserted by \(\text{fMet} - \text{tRNA}_\text{f}^\text{Met}\) at the beginning of every polypeptide chain synthesized by E. coli, but it is usually clipped off after translation is complete. When an in-frame AUG is encountered during translation elongation, a non-formylated methionine is inserted by a regular Met-tRNA^Met.

In E. coli mRNA, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. Guanosine triphosphate (GTP), which is a purine nucleotide triphosphate, acts as an energy source during translation—both at the start of elongation and during the ribosome’s translocation.

In eukaryotes, a similar initiation complex forms, comprising mRNA, the 40S small ribosomal subunit, IFs, and nucleoside triphosphates (GTP and ATP). The charged initiator tRNA, called Met-tRNA_i, does not bind fMet in eukaryotes, but is distinct from other Met-tRNAs in that it can bind IFs.

Instead of depositing at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5' end of the mRNA. A cap-binding protein (CBP) and several other IFs assist the movement of the ribosome to the 5' cap. Once at the cap, the initiation complex tracks along the mRNA in the 5' to 3' direction, searching for the AUG start codon. Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case. According to Kozak’s rules, the nucleotides around the AUG indicate whether it is the correct start codon. Kozak’s rules state that the following consensus sequence must appear around the AUG of vertebrate genes: 5'-gccRccAUGG-3'. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.

Once the appropriate AUG is identified, the other proteins and CBP dissociate, and the 60S subunit binds to the complex of Met-tRNA_i, mRNA, and the 40S subunit. This step completes the initiation of translation in eukaryotes.

Translation, Elongation, and Termination

In prokaryotes and eukaryotes, the basics of elongation are the same, so we will review elongation from the perspective of E. coli. The 50S ribosomal subunit of E. coli consists of three compartments: the A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one exception to this assembly line of tRNAs: in E. coli, \(\text{fMet} - \text{tRNA}_\text{f}^\text{Met}\) is capable of entering the P site directly without first entering the A site. Similarly, the eukaryotic Met-tRNA_i, with help from other proteins of the initiation complex, binds directly to the P site. In both cases, this creates an initiation complex with a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

During translation elongation, the mRNA template provides specificity. As the ribosome moves along the mRNA, each mRNA codon comes into register, and specific binding with the corresponding charged tRNA anticodon is ensured. If mRNA were not present in the elongation complex, the ribosome would bind tRNAs nonspecifically.

Elongation proceeds with charged tRNAs entering the A site and then shifting to the P site followed by the E site with each single-codon “step” of the ribosome. Ribosomal steps are induced by conformational changes that advance the ribosome by three bases in the 3' direction. The energy for each step of the ribosome is donated by an elongation factor that hydrolyzes GTP. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based enzyme that is integrated into the 50S ribosomal subunit. The energy for each peptide bond formation is derived from GTP hydrolysis, which is catalyzed by a separate elongation factor. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled (Figure \(\PageIndex{2}\)). Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid protein can be translated in just 10 seconds.

Art Connection

Figure \(\PageIndex{2}\): Translation begins when an initiator tRNA anticodon recognizes a codon on mRNA. The large ribosomal subunit joins the small subunit, and a second tRNA is recruited. As the mRNA moves relative to the ribosome, the polypeptide chain is formed. Entry of a release factor into the A site terminates translation and the components dissociate.

Many antibiotics inhibit bacterial protein synthesis. For example, tetracycline blocks the A site on the bacterial ribosome, and chloramphenicol blocks peptidyl transfer. What specific effect would you expect each of these antibiotics to have on protein synthesis?

Tetracycline would directly affect:

1. tRNA binding to the ribosome
2. ribosome assembly
3. growth of the protein chain

Chloramphenicol would directly affect

1. tRNA binding to the ribosome
2. ribosome assembly
3. growth of the protein chain

Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

Protein Folding, Modification, and Targeting

During and after translation, individual amino acids may be chemically modified, signal sequences may be appended, and the new protein “folds” into a distinct three-dimensional structure as a result of intramolecular interactions. A signal sequence is a short tail of amino acids that directs a protein to a specific cellular compartment. These sequences at the amino end or the carboxyl end of the protein can be thought of as the protein’s “train ticket” to its ultimate destination. Other cellular factors recognize each signal sequence and help transport the protein from the cytoplasm to its correct compartment. For instance, a specific sequence at the amino terminus will direct a protein to the mitochondria or chloroplasts (in plants). Once the protein reaches its cellular destination, the signal sequence is usually clipped off.

Many proteins fold spontaneously, but some proteins require helper molecules, called chaperones, to prevent them from aggregating during the complicated process of folding. Even if a protein is properly specified by its corresponding mRNA, it could take on a completely dysfunctional shape if abnormal temperature or pH conditions prevent it from folding correctly.

Summary

The players in translation include the mRNA template, ribosomes, tRNAs, and various enzymatic factors. The small ribosomal subunit forms on the mRNA template either at the Shine-Dalgarno sequence (prokaryotes) or the 5' cap (eukaryotes). Translation begins at the initiating AUG on the mRNA, specifying methionine. The formation of peptide bonds occurs between sequential amino acids specified by the mRNA template according to the genetic code. Charged tRNAs enter the ribosomal A site, and their amino acid bonds with the amino acid at the P site. The entire mRNA is translated in three-nucleotide “steps” of the ribosome. When a nonsense codon is encountered, a release factor binds and dissociates the components and frees the new protein. Folding of the protein occurs during and after translation.

Art Connections

Figure \(\PageIndex{2}\): Many antibiotics inhibit bacterial protein synthesis. For example, tetracycline blocks the A site on the bacterial ribosome, and chloramphenicol blocks peptidyl transfer. What specific effect would you expect each of these antibiotics to have on protein synthesis?

Tetracycline would directly affect:

1. tRNA binding to the ribosome
2. ribosome assembly
3. growth of the protein chain

Chloramphenicol would directly affect

1. tRNA binding to the ribosome
2. ribosome assembly
3. growth of the protein chain

Answer

Tetracycline: a; Chloramphenicol: c.

Glossary

aminoacyl tRNA synthetase

enzyme that “charges” tRNA molecules by catalyzing a bond between the tRNA and a corresponding amino acid

initiator tRNA

in prokaryotes, called \(\text{tRNA}_\text{f}^\text{Met}\); in eukaryotes, called tRNA_i; a tRNA that interacts with a start codon, binds directly to the ribosome P site, and links to a special methionine to begin a polypeptide chain

Kozak’s rules

determines the correct initiation AUG in a eukaryotic mRNA; the following consensus sequence must appear around the AUG: 5’-GCC(purine)CCAUGG-3’; the bolded bases are most important

peptidyl transferase

RNA-based enzyme that is integrated into the 50S ribosomal subunit and catalyzes the formation of peptide bonds

polysome

mRNA molecule simultaneously being translated by many ribosomes all going in the same direction

Shine-Dalgarno sequence

(AGGAGG); initiates prokaryotic translation by interacting with rRNA molecules comprising the 30S ribosome

signal sequence

short tail of amino acids that directs a protein to a specific cellular compartment

start codon

AUG (or rarely, GUG) on an mRNA from which translation begins; always specifies methionine