4 Translation

Learning Objectives

Upon completing this chapter, you will be able to:

• Explain the “genetic code” and how nucleotide triplets (codons) in mRNA specify amino acids.
• Describe the properties of the genetic code, including start codons, and stop codons.
• Identify and describe the key components of the protein synthesis machinery: mRNA, ribosomes (and their subunits), and transfer RNAs (tRNAs), including their specific roles.
• Outline and describe the three main stages of translation in bacteria: initiation, elongation, and termination.
• Identify the key sites on the ribosome (A, P, and E sites) and their functions during elongation.

Introduction: Decoding the Message

The synthesis of proteins is a highly energy-consuming process, yet proteins are fundamental to life, accounting for more mass than any other macromolecule in living organisms. They are the workhorses of the cell, performing a vast array of functions, from catalyzing biochemical reactions (as enzymes) to providing1 structural support. The process of translation, or protein synthesis, is the second critical part of gene expression. It involves the decoding of a messenger RNA (mRNA) message by a ribosome into a specific sequence of amino acids, forming a polypeptide product.

The Genetic Code: The Language of Life

Translation converts nucleotide-based genetic information from an mRNA template into the “language” of amino acids to build a protein. A protein sequence is typically composed of 20 commonly occurring amino acids.

• Codons: Each amino acid within the mRNA is defined by a triplet of nucleotides called a codon. For example, an mRNA molecule contains codons that will encode for specific amino acids.
• The Genetic Code: The specific relationship between an mRNA codon and its corresponding amino acid is defined by the genetic code.
• Combinations & Degeneracy: With four different nucleotides (A, U, G, C) and three positions in each codon, there are 64 possible codon combinations. This is more than the 20 common amino acids. Consequently, most amino acids are encoded by more than one codon. This redundancy in the genetic code is termed degeneracy.
• Wobble Position: Typically, the first two nucleotides in a codon are crucial for determining the specific amino acid. The third nucleotide, known as the wobble position, is often less critical. A change in the nucleotide at this third position may still result in the incorporation of the same amino acid.
• Start Codon: The codon AUG has a dual function. It codes for the amino acid methionine (or N-formylmethionine in bacteria during initiation) and also typically serves as the start codon, signaling the beginning of protein synthesis.
• Stop Codons (Nonsense Codons): Three of the 64 codons – UAA, UAG, and UGA – do not code for any amino acid. Instead, they act as stop codons, signaling the termination of protein synthesis and causing the release of the completed polypeptide from the translation machinery.
• Reading Frame: The reading frame refers to how nucleotides in an mRNA are grouped into codons for translation. The reading frame is critically established by the AUG start codon, usually found near the 5’ end of the mRNA. Every set of three nucleotides following this start codon constitutes a codon in the mRNA message.
• Near Universality: The genetic code is remarkably consistent across nearly all species, from bacteria to humans. This near universality provides strong evidence for a common evolutionary origin of all life on Earth.
  

  

The Protein Synthesis Machinery

The intricate process of translation requires the coordinated action of many molecular components beyond the mRNA template. These include ribosomes, transfer RNAs (tRNAs), and various enzymatic factors. While the exact composition can vary between organisms (e.g., bacterial vs. eukaryotic ribosomes), the fundamental structures and functions are highly conserved.

Ribosomes: The Protein Factories

A ribosome is a complex macromolecular machine responsible for protein synthesis. It’s composed of ribosomal RNA (rRNA) molecules and numerous distinct polypeptides.

• Composition: Mature rRNAs constitute about 50% of a ribosome’s mass. Some rRNAs possess catalytic activity and are called ribozymes (e.g., peptidyl transferase activity).
• Prokaryotic vs. Eukaryotic Ribosomes:
  • Prokaryotes have 70S ribosomes, which are composed of a small 30S subunit (containing 16S rRNA) and a large 50S subunit.
  • Eukaryotes have larger 80S ribosomes in their cytoplasm and on the rough endoplasmic reticulum, consisting of a small 40S subunit (containing 18S rRNA) and a large 60S subunit. Eukaryotes also have 70S ribosomes in their mitochondria and chloroplasts, reflecting their prokaryotic ancestry.

• Assembly and Function: Ribosomes typically exist as separate small and large subunits in the cytoplasm when not actively synthesizing proteins. They associate during the initiation of translation.
• The small ribosomal subunit is primarily responsible for binding the mRNA template.
• The large ribosomal subunit binds tRNAs and catalyzes the formation of peptide bonds to link amino acids into a polypeptide chain.

Transfer RNAs (tRNAs): The Adaptor Molecules

Transfer RNAs (tRNAs) are crucial adaptor molecules that bridge the language of nucleic acids (codons in mRNA) and the language of proteins (amino acids).

• Function: Each tRNA type specifically binds to a particular mRNA codon and carries the corresponding amino acid to be added to the growing polypeptide chain. Bacterial species typically have between 60 and 90 different types of tRNAs.
• Structure: Mature tRNAs are relatively small RNA molecules (70-90 nucleotides) that fold into a characteristic three-dimensional L-shape due to intramolecular hydrogen bonding between complementary base sequences. This structure features two key regions:
  • The amino acid binding end: A sequence at the 3’ end of the tRNA molecule where the specific amino acid is attached.
  • The anticodon loop: Contains a three-nucleotide sequence called the anticodon, which is complementary to and base-pairs with a specific mRNA codon.
    

    

The Mechanism of Protein Synthesis

Translation is a dynamic process generally divided into three phases: initiation, elongation, and termination. While the fundamental steps are similar in prokaryotes and eukaryotes, we will focus on translation in E. coli, a representative prokaryote, noting key differences.

1. Initiation: Getting Started

The initiation of protein synthesis involves the assembly of the translation machinery at the start codon of the mRNA.

• Formation of the Initiation Complex in E. coli:

1. The small ribosomal subunit binds to the mRNA template.
2. A special initiator tRNA carrying N-formyl-methionine  binds to the AUG start codon on the mRNA. N-formyl-methionine (fMet) is the modified amino acid that begins every polypeptide chain synthesized in E. coli.
3. Several initiation factors (proteins) assist in the correct assembly of this complex.
4. Once these components are assembled, the large (50S) ribosomal subunit binds to the initiation complex, forming the complete, functional 70S ribosome.

2. Elongation: Building the Polypeptide Chain

Once the initiation complex is formed with the initiator tRNA in place, the ribosome is ready to extend the polypeptide chain. Elongation involves a cyclical process of adding amino acids one by one. The intact ribosome has three key sites for tRNA interaction:

• A (aminoacyl) site: The entry point for incoming charged aminoacyl-tRNAs (tRNAs carrying their specific amino acid).
• P (peptidyl) site: Holds the tRNA carrying the growing polypeptide chain. (During initiation, the fMet-tRNA^fMet or Met-tRNA_i directly enters the P site, leaving the A site free for the next tRNA).
• E (exit) site: The site from which dissociated (uncharged) tRNAs leave the ribosome after they have donated their amino acid.

The Steps of Elongation (repeated for each amino acid added):

1. Codon Recognition & tRNA Entry: A charged tRNA with an anticodon complementary to the mRNA codon exposed in the A site enters and binds. This step requires GTP hydrolysis, facilitated by elongation factors.
2. Peptide Bond Formation: The amino acid attached to the tRNA in the P site (or fMet for the first amino acid) is uncoupled from its tRNA and linked via a peptide bond to the amino group of the amino acid attached to the tRNA in the A site. This reaction is catalyzed by peptidyl transferase, an enzymatic activity inherent to the rRNA (a ribozyme) within the large ribosomal subunit. The growing polypeptide chain is thus transferred from the tRNA in the P site to the tRNA in the A site.
3. Translocation: The ribosome moves exactly one codon (three nucleotides) along the mRNA in the 5’ to 3’ direction. This step also requires GTP hydrolysis and is driven by other elongation factors.

• The tRNA that was in the P site (now uncharged) moves to the E site and is subsequently released from the ribosome, free to be recharged with another amino acid.
• The tRNA that was in the A site (now carrying the growing polypeptide chain) moves into the P site.
• The A site is now vacant and positioned over the next codon on the mRNA, ready to accept the next charged tRNA.

This cycle of charged tRNA entry, peptide bond formation, and translocation repeats, adding amino acids to the polypeptide chain. In E. coli, this process is remarkably rapid, with each amino acid added in about 0.05 seconds, meaning a 200-amino acid protein can be synthesized in just 10 seconds. As elongation continues, the growing peptide is continually transferred to the A-site tRNA, the ribosome moves along the mRNA, and new tRNAs enter.

3. Termination: Releasing the Product

Translation ends when a stop codon (UAA, UAG, or UGA) on the mRNA enters the A site of the ribosome.

1. Recognition of Stop Codon: There are no tRNAs with anticodons complementary to stop codons. Instead, proteins called release factors recognize these nonsense codons when they align with the A site.
2. Polypeptide Release: The binding of a release factor causes the peptidyl transferase to catalyze the addition of a water molecule to the P-site tRNA, detaching the completed polypeptide chain from the tRNA. The newly made polypeptide is then released from the ribosome.
3. Ribosome Dissociation: The ribosomal subunits (small and large) dissociate from the mRNA and from each other. They are then free to be recycled and participate in another round of translation initiation on a new mRNA molecule.

Chapter Summary

Translation is the vital process of protein synthesis, where the genetic information encoded in mRNA codons is decoded by ribosomes to produce a specific polypeptide chain. The genetic code dictates which amino acid corresponds to each three-nucleotide codon, with AUG typically serving as the start codon (methionine/fMet) and UAA, UAG, or UGA acting as stop codons.

The machinery for translation includes:

• mRNA: Carries the genetic blueprint from DNA.
• Ribosomes (70S in prokaryotes, 80S in eukaryotic cytoplasm): Composed of rRNA and proteins, with small and large subunits. They provide the sites (A, P, E) for tRNA binding and catalyze peptide bond formation.
• Transfer RNAs (tRNAs): Act as adaptors, each carrying a specific amino acid and possessing an anticodon complementary to an mRNA codon. tRNAs are charged with their correct amino acid by aminoacyl tRNA synthetases.

Translation occurs in three stages:

1. Initiation: The small ribosomal subunit binds mRNA (at the Shine-Dalgarno sequence in E. coli), the initiator tRNA (carrying fMet in E. coli) binds to the start codon in the P site, and the large subunit joins to form a functional ribosome.
2. Elongation: Charged tRNAs enter the A site, a peptide bond is formed between the amino acid in the A site and the growing polypeptide in the P site (catalyzed by peptidyl transferase), and the ribosome translocates one codon along the mRNA, shifting tRNAs through the P and E sites.
3. Termination: When a stop codon enters the A site, release factors trigger the release of the completed polypeptide, and the ribosomal complex dissociates.

In prokaryotes, translation can occur concurrently with transcription, allowing for rapid protein production. This intricate process ensures the accurate synthesis of proteins that carry out nearly all cellular functions.