Cyclohexamide(CHX) as a translation elongation inhibitor in Eukaryotes

Cycloheximide is an inhibitor of protein biosynthesis in eukaryotic organisms, produced by the bacterium Streptomyces griseus. Cycloheximide exerts its effect by interfering with the translocation step in protein synthesis (movement of two tRNA molecules and mRNA in relation to the ribosome) thus blockingtranslational elongation. Cycloheximide is widely used in biomedical research to inhibit protein synthesis in eukaryotic cells studied in vitro (i.e. outside of organisms). It is inexpensive and works rapidly. Its effects are rapidly reversed by simply removing it from the culture medium.

Due to significant toxic side effects, including DNA damage, teratogenesis, and other reproductive effects (including birth defects and toxicity to sperm^[1]), cycloheximide is generally used only in in vitro research applications, and is not suitable for human use as a therapeutic compound. Although it has been used as a fungicide in agricultural applications, this application is now decreasing as the health risks have become better understood.

Because cycloheximide is degraded by alkali (pH > 7), decontamination of work surfaces and containers can be achieved by washing with a non-harmful alkali solution such as soap.

Cycloheximide can be used as an experimental tool in molecular biology to determine the half-life of a protein. Treating cells with cycloheximide in a time-course experiment followed by Western blotting of the cell lysates for the protein of interest can show differences in protein half-life. Cycloheximide treatment provides the ability to observe the half-life of a protein without confounding contributions from transcription or translation.

It is used as a plant growth regulator to stimulate ethylene production. It is used as a rodenticide and other animal pesticide. It is also used in media to detect unwanted bacteria by suppressing yeasts and molds in beer fermentation.

The translational elongation freezing properties of Cycloheximide are also used for ribosome profiling / translational profiling. Translation is halted via the addition of cycloheximide, and the DNA/RNA in the cell is then nuclease treated. The ribosome-bound parts of RNA can then be sequenced.

cited from wiki

Cycloheximide (CHX)

Lactimidomycin (LTM)

Tilman Schneider-Poetsch, 

Nature Chemical Biology

6, 209–217 (2010)

Tilman Schneider-Poetsch, 

Nature Chemical Biology

6, 209–217 (2010)

Tilman Schneider-Poetsch, 

Nature Chemical Biology

6, 209–217 (2010)

" ..... 

In this model, 
CHX and LTM have a largely similar mechanism through binding to the E-site of the 60S ribosome. The binding of CHX and LTM to the E-site blocks eEF2-mediated tRNA translocation. This model explains the similar effects of CHX and LTM on translational elongation and tripeptide formation. A difference between CHX and LTM lies in the ability of CHX to bind the E-site together with the E-site tRNA while the larger LTM occludes deacylated tRNA from the E-site. Thus, binding of CHX to the E-site alone does not affect translocation, whereas occupation of the E-site by both CHX and deacylated tRNA leads to an arrest of the ribosome on the second codon. This is in agreement with the observation by others that CHX allows for two rounds of translocation on a CrPV IRES template, because the cricket paralysis virus element initiates translation without initiator tRNA and begins translation from the A-site³. Consequently it takes two translocation events before deacylated tRNA reaches the E-site. While LTM binds to the same site, its sheer size occludes deacylated tRNA. Thus LTM blocks the very first round of elongation and prevents the ribosome from leaving the start site. Unlike LTM, the smaller CHX presumably can also bind to actively translocating ribosomes, providing a plausible explanation for the distinct polysome profiles for LTM and CHX. In contrast to CHX, the mutually exclusive binding of LTM and deacylated tRNA to the E-site leads to preferential binding of LTM to an empty E-site, allowing dipeptide formation but blocking the translocation of the newly formed deacylated initiating tRNA from the P-site to the E-site, thus stalling the ribosome at the AUG start codon. Once elongation has been initiated and the E-site is occupied by deacylated tRNA, it will be more difficult for LTM to gain access to the E-site, leading to polysome depletion. Unlike LTM, CHX can interrupt translation elongation at any time, as its binding to the E-site is independent of the occupancy of the E-site by deacylated tRNA. Thus, its polysome profile does not significantly differ from that of an untreated cell. We note that this model does not seem to account for the effect of CHX on the eEF2-mediated translocation assay indirectly measured by phenylalanyl puromycin formation at first sight. However, there was plenty of deacylated tRNA present in the translocation assay, whose co-occupation of the E-site may explain the inhibition seen by CHX ........"

cited from Tilman Schneider-Poetsch, 

Nature Chemical Biology

6, 209–217 (2010)