Cycloheximide is a compound produced naturally by the bacterium Streptomyces griseus. Its primary function is as a potent inhibitor of protein synthesis. This action makes it a valuable tool for researchers in the fields of cell biology and molecular science. It allows for the specific and rapid halting of protein production within eukaryotic cells, providing a window into various cellular functions.
The Target: Eukaryotic Protein Synthesis Machinery
Protein synthesis, also known as translation, is the process by which cells build proteins. In eukaryotic cells, which include those of plants, animals, and fungi, this operation is carried out by molecular machines called ribosomes. The ribosome reads instructions from a messenger RNA (mRNA) molecule, a template copied from the cell’s DNA. Each set of three letters on the mRNA, called a codon, specifies a particular amino acid.
These amino acids are brought to the ribosome by transfer RNA (tRNA) molecules. The ribosome itself is composed of a large 60S subunit and a small 40S subunit. Within the assembled ribosome, there are three docking locations: the A-site (aminoacyl), P-site (peptidyl), and E-site (exit). A new tRNA enters the A-site, a peptide bond adds its amino acid to the chain in the P-site, and the empty tRNA moves to the E-site before release. The entire assembly then shifts one codon down the mRNA in a process called translocation.
Cycloheximide’s Inhibitory Action
Cycloheximide halts protein synthesis by directly targeting the ribosome during the elongation phase of translation. Its specific binding partner is the E-site located on the large 60S ribosomal subunit. By lodging itself within this exit site, cycloheximide creates a physical barricade that interferes with the step of translocation.
The binding of cycloheximide to the E-site prevents the ribosome from moving along the mRNA template, effectively jamming the entire protein synthesis machinery. The tRNA in the P-site cannot move to the E-site, and a new tRNA cannot enter the A-site because the ribosome is frozen. This action stops the polypeptide chain from growing any further.
The effect is rapid, shutting down the production of nearly all proteins within the cell. This inhibition continues as long as the compound is present. However, for many cell types, its effects can be reversed by simply washing the cycloheximide out of the cellular environment, allowing the ribosomes to resume their function.
Selective Impact on Eukaryotic Cells
A defining characteristic of cycloheximide is its high degree of selectivity. It is a powerful inhibitor of protein synthesis in eukaryotic organisms, including animals, plants, and fungi, but has no effect on the protein synthesis machinery of prokaryotic cells like bacteria. This specificity is the reason it can be used as an antifungal agent without harming beneficial bacteria.
The basis for this selective impact lies in the structural differences between the ribosomes of these different life forms. Eukaryotic cells possess 80S ribosomes, which are composed of the 60S and 40S subunits. In contrast, prokaryotic cells have smaller 70S ribosomes, made from 50S and 30S subunits.
These differences in size and composition are most significant in the large ribosomal subunit. The precise three-dimensional structure of the E-site on the eukaryotic 60S subunit creates a perfect binding pocket for the cycloheximide molecule. This specific binding site is absent or structurally different in the prokaryotic 50S subunit, meaning cycloheximide has no target to interact with in bacteria.
Applications as a Research Tool
The ability of cycloheximide to rapidly and specifically halt protein synthesis in eukaryotes makes it a useful tool in scientific research. One of its primary uses is in studying the lifespan, or half-life, of proteins. By treating cells with cycloheximide, researchers can stop the production of new proteins and then measure how quickly the existing ones are degraded by the cell over time.
Scientists also use cycloheximide to determine if a particular cellular process requires the synthesis of new proteins. For instance, if a researcher wants to know whether programmed cell death (apoptosis) needs to produce new proteins, they can treat the cells with cycloheximide. If apoptosis is blocked, it indicates that the synthesis of one or more new proteins is a necessary step.
This research application extends to complex biological systems, such as the study of memory and learning. Experiments involving the administration of cycloheximide to specific brain regions have helped demonstrate that the consolidation of long-term memories requires the synthesis of new proteins in neurons. Blocking this synthesis can prevent new memories from being stored permanently.
Beyond the laboratory, cycloheximide’s selective action led to its use as a fungicide in agriculture to protect crops from fungal infections. It has also been used to help isolate bacteria from environmental samples by suppressing the growth of competing fungi and yeasts. Its use has been limited due to toxicity concerns.