Puromycin is an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. It is a potent inhibitor of protein synthesis that stops the elongation of polypeptide chains during translation. This action halts cell growth and has made it a tool in biomedical research.
Structural Mimicry of tRNA
The effectiveness of puromycin stems from its structural similarity to a molecule involved in protein synthesis called transfer RNA (tRNA). In a functioning cell, tRNA molecules are responsible for transporting specific amino acids—the building blocks of proteins—to the ribosome. Each tRNA molecule has a distinct end that attaches to an amino acid; this complex is known as an aminoacyl-tRNA. The ribosome then adds this amino acid to the growing protein chain.
Puromycin’s structure closely resembles the 3′ end of an aminoacyl-tRNA, particularly that of tyrosyl-tRNA. Due to this molecular mimicry, the ribosome is tricked into accepting puromycin as if it were a natural component, which is the basis of its inhibitory action.
Interaction with the Ribosome
Once inside the cell, puromycin’s structural likeness to an aminoacyl-tRNA allows it to enter a specific location on the ribosome known as the aminoacyl site, or A-site. The A-site is the ribosome’s “landing pad” for incoming tRNA molecules carrying the next amino acid to be added to the polypeptide chain. The ribosome’s catalytic core, the peptidyl transferase center, is responsible for forming the peptide bonds that link amino acids together.
The peptidyl transferase center performs its designated function and attempts to create a peptide bond. The ribosome catalyzes a reaction that links the growing polypeptide chain, which is held in the adjacent peptidyl site (P-site), to the amino group of the puromycin molecule in the A-site.
The ribosome mistakenly attaches the partially built protein to the puromycin molecule, forming what is called a peptidyl-puromycin product. This event terminates the protein’s growth and marks the point of no return for its synthesis.
Causing Premature Chain Termination
The newly formed peptidyl-puromycin molecule is unable to continue the translation cycle. This is because puromycin possesses a stable amide bond, which differs from the less stable ester bond found in a normal aminoacyl-tRNA.
This chemical difference prevents the ribosome from moving the peptidyl-puromycin from the A-site to the P-site, a step known as translocation. Furthermore, the puromycin molecule lacks the necessary chemical structures to allow for the attachment of any subsequent amino acids. The ribosome is effectively stuck with a capped-off, incomplete protein that it cannot extend or process further.
The ultimate result is the premature release of this truncated and non-functional protein from the ribosome. This action poisons the translation machinery, leading to an accumulation of useless protein fragments within the cell. This disruption of protein production is what underlies puromycin’s potent biological effects.
Use as a Laboratory Selection Agent
Beyond its use in studying the mechanics of protein synthesis, puromycin is a widely used tool in molecular biology for genetic selection. In research settings, scientists often need to grow cells that have successfully incorporated foreign DNA, typically in the form of a circular piece of DNA called a plasmid. These plasmids are often engineered to carry a gene of interest alongside a puromycin resistance gene, such as the puromycin N-acetyltransferase (pac) gene.
When this plasmid is introduced into a population of cells, only a fraction of them will successfully take it up. To isolate these modified cells, scientists add puromycin to the cell culture medium. The cells that did not incorporate the plasmid are unable to neutralize the antibiotic, and their protein synthesis is shut down, leading to rapid cell death.
In contrast, the cells that have the plasmid can express the resistance gene. The enzyme produced by the pac gene, for example, inactivates puromycin by chemically modifying its reactive amino group. This allows these cells to survive and proliferate in the presence of the antibiotic, providing researchers with a pure population of genetically engineered cells.
Limitations Due to Non-Selectivity
While a powerful laboratory tool, puromycin is not used as a clinical antibiotic in humans due to its lack of selectivity. It inhibits protein synthesis in both prokaryotic cells, like bacteria, and eukaryotic cells, such as those in humans. This is because the structure and function of the ribosome are highly conserved across different domains of life.
The ribosome’s A-site and peptidyl transferase center in both types of cells are vulnerable to puromycin’s deceptive mechanism. Consequently, the antibiotic is highly toxic to human cells, not just invading pathogens. Administering it to a patient would cause widespread inhibition of protein synthesis in the host’s own tissues, leading to severe side effects.
This contrasts with many clinically useful antibiotics, like penicillin, which target structures unique to bacteria, such as the cell wall. Because human cells lack a cell wall, penicillin can kill bacteria without harming the patient. Puromycin’s inability to distinguish between cell types confines its application to the research laboratory.