Biology » Genes and Proteins » Ribosomes and Protein Synthesis

# The Mechanism of Protein Synthesis

## The Mechanism of Protein Synthesis

As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes. Here we’ll explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.

## Initiation of Translation

Protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors (IFs; IF-1, IF-2, and IF-3), and a special initiator tRNA, called $$\text{t}\text{R}\text{N}{\text{A}}_{\text{f}}^{\text{M}\text{e}\text{t}}$$. 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 $$\text{f}\text{M}\text{e}\text{t}-\text{t}\text{R}\text{N}{\text{A}}_{\text{f}}^{\text{M}\text{e}\text{t}}$$ 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-tRNAMet.

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-tRNAi, 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-tRNAi, 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, $$\text{f}\text{M}\text{e}\text{t}-\text{t}\text{R}\text{N}{\text{A}}_{\text{f}}^{\text{M}\text{e}\text{t}}$$ is capable of entering the P site directly without first entering the A site. Similarly, the eukaryotic Met-tRNAi, 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 (see the figure below). 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

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

Tetracycline: tRNA binding to the ribosome;
Chloramphenicol: 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.

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