Inside cells the molecule transfer RNA (t-RNA) (1) is a key factor in protein sy
ID: 213467 • Letter: I
Question
Inside cells the molecule transfer RNA (t-RNA) (1) is a key factor in protein synthesis. Its structure involves a long chain of nucleotides, terminated by an adenosine nucleotide unit which is esterified at the 3'-OH group of the ribose sugar ring. Puromycin (2) is a drug which blocks protein synthesis in the cell. NT SN CH2 0 NH OH CH-NH CH-NH2 CH2 CH (a) Puromycin (2) is considered an isostere of transfer RNA (1). Eplain clearly why this so. (b) A nitrogen and oxygen in puromycin are methylated. How might this efffect the ability of the drug to be delivered to its target? c) Suggest another isostere which might possibly be even more effective han puromycin.Explanation / Answer
A)
Puromycin is an aminonucleoside antibiotic, derived from the Streptomyces alboniger bacterium, that causes premature chain termination during translation taking place in the ribosome. Part of the molecule resembles the 3' end of the aminoacylated tRNA, it enters the A site and transfers to the growing chain, causing the formation of a puromycylated nascent chain and premature chain release. The exact mechanism of action is unknown at this time but the 3' position contains an amide linkage instead of the normal ester linkage of tRNA. That makes the molecule much more resistant to hydrolysis and stops the ribosome.
Puromycin is selective for either prokaryotes or eukaryotes.
Also of note, puromycin is critical in mRNA display; in this reaction, a puromycin molecule is chemically attached to the end of an mRNA template, which is then translated into protein. The puromycin can then form a covalent link to the growing peptide chain allowing the mRNA to be physically linked to its translational product.
Antibodies that recognize puromycylated nascent chains can also be used to purify newly synthesized polypeptides and to visualize the distribution of actively translating ribosomes by immunofluorescence.
B) One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood–brain barrier. As Pglycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five Pglycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4µg/mL puromycin for the first 2days of culture or 3µg/mL puromycin for the first 3days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycintreated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75×106cm/s) and a high electrical resistance (up to 500×cm2). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood–brain barrier for cellular and molecular biology studies as well as pharmacological investigations.
c) Sites of cycloheximide action on protein synthesis wereexamined using a cell-free system prepared from rat liver.If all amino acids or aminoacyl transfer RNA were presentat the start of incubation, the system appeared to incor-porate W-leucine mainly by elongation of peptide chains.Under these conditions, high dose levels of cycloheximidewere necessary in order to inhibit incorporation extensively.The inhibition could be prevented by raising the glutathionecontent of the reaction mixture, and particularly by prelimi-nary incubation of a mixture of transferase I and II withhigh concentrations of glutathione before adding theseenzymes to the system. Other sulfhydryl compounds werealso effective in protecting against cycloheximide. It hasbeen concluded that the inhibitory action of cycloheximideon peptide chain elongation involves inactivation of trans-ferase II, an enzyme known to have a sulfhydryl requirement.