Prokaryotic Translation

Translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between bacterial and eukaryotic translation.

Initiation

The initiation of 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 that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-methionine (fMet-tRNAfMet) (Figure 1). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli. In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.

In eukaryotes, initiation complex formation is similar, with the following differences:

  • The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi
  • Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5′ cap of the eukaryotic mRNA, then tracks along the mRNA in the 5′ to 3′ direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.
Diagram showing translation. At the start codon of the mRNA (AUG) the following attach: a tRNA with the anticodon UAC and containing the first amino acid, the large ribosomal subunit (a dome) and the small ribosomal subunit (a flat oval). During initiation, translational complex forms, and tRNA brings the first amino acid in polypeptide chain to bind to start codon om mRNA. At this point the tRNA is attached to the middle binding site (P) of the ribosome. The 3 sites from left to right are E, P, A. During elongation, tRNAs bring amino acids one by one to add to polypeptide chain. In the diagram, a tRNA with a long chain of circles is in the P site, a tRNA with a single circle is in the A site, and a tRNA without any circles is leaving from the E site. During termination, release factor recognizes stop codon, translational complex dissociates, and complete polypeptide is released. In the diagram a tRNA with a long strand is attached to the P site and a release factor (red shape) is attached to the stop codon in the mRNA which is now under the A site. Next the completed polypeptide leaves and all the other components dissociate from each other.

Figure 1. Translation in bacteria begins with the formation of the initiation complex, which includes the small ribosomal subunit, the mRNA, the initiator tRNA carrying N-formyl-methionine, and initiation factors. Then the 50S subunit binds, forming an intact ribosome.

Elongation

In prokaryotes and eukaryotes, the basics of elongation of translation are the same. In E. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites: 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 notable exception to this assembly line of tRNAs: During initiation complex formation, bacterial fMet−tRNAfMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3′ direction. 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 ribozyme that is integrated into the 50S ribosomal subunit. 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. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. 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.

Termination

The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation init iation complex.

In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are illustrated in Figure 2 and listed in Table 1.

a) Diagram of prokaryotic cell with a plasma membrane on the outside. The DNA is in the cytoplasm and the mRNA is being copied at the same time that ribosomes are building proteins of the developing mRNA. B) Diagram of a eukaryotic cell with a plasma membrane an a nucleus. The DNA is in the nucleus and pre-mRNA is made during transcription; this is then process into mature mRNA. The mature mRNA then leaves the nucleus and enters the cytoplasm where translation takes place. This is when ribosomes bind to the mRNA and make proteins.

Figure 2. (a) In prokaryotes, the processes of transcription and translation occur simultaneously in the cytoplasm, allowing for a rapid cellular response to an environmental cue. (b) In eukaryotes, transcription is localized to the nucleus and translation is localized to the cytoplasm, separating these processes and necessitating RNA processing for stability.

Table 1. Comparison of Translation in Bacteria Versus Eukaryotes
Property Bacteria Eukaryotes
Ribosomes 70S

  • 30S (small subunit) with 16S rNA subunit
  • 50S (large subunit) with 5S and 23S rRNA subunits
80S

  • 40S (small subunit) with 18S rRNA subunit
  • 60S (large subunit) with 5S, 5.8S, and 28S rRNA subunits
Amino acid carried by initiator tRNA fMet Met
Shine-Dalgarno sequence in mRNA Present Absent
Simultaneous transcription and translation Yes No