The science of puromycin: From studies of ribosome function to applications in biotechnology

Abstract

Puromycin is a naturally occurring aminonucleoside antibiotic that inhibits protein synthesis by ribosome-catalyzed incorporation into the C-terminus of elongating nascent chains, blocking further extension and resulting in premature termination of translation. It is most commonly known as a selection marker for cell lines genetically engineered to express a resistance transgene, but its additional uses as a probe for protein synthesis have proven invaluable across a wide variety of model systems, ranging from purified ribosomes and cell-free translation to intact cultured cells and whole animals. Puromycin is comprised of a nucleoside covalently bound to an amino acid, mimicking the 3' end of aminoacylated tRNAs that participate in delivery of amino acids to elongating ribosomes. Both moieties can tolerate some chemical substitutions and modifications without significant loss of activity, generating a diverse toolbox of puromycin-based reagents with added functionality, such as biotin for affinity purification or fluorophores for fluorescent microscopy detection. These reagents, as well as anti-puromycin antibodies, have played a pivotal role in advancing our understanding of the regulation and dysregulation of protein synthesis in normal and pathological processes, including immune response and neurological function. This manuscript reviews the current state of puromycin-based research, including structure and mechanism of action, relevant derivatives, use in advanced methodologies and some of the major insights generated using such techniques both in the lab and the clinic.

Keywords: Nascent polypeptide chains; O-propargyl-puromycin (OPP); PUNCH-P; Protein synthesis; Puromycin; Ribosome; SUnSET; Translation; mRNA display; puro-PLA.

Fig. 1

Puromycin structure and mechanism of action. (A) Chemical structures of the 3′ end of an aminoacylated tyrosyl-tRNA (left) and puromycin (right). The different bonds between the nucleoside and amino acid moieties are shown in pink. (B) Basic mechanism of puromycin action. During translation elongation, aa-tRNA enters the A-site and accepts the nascent polypeptide chain from the peptidyl-tRNA in the P-site. Following translocation, the A-site becomes available to accommodate the next aa-tRNA (top). Like aa-tRNA, puromycin can enter the A-site and accept the nascent chain. This results in translation termination, ribosome disassembly and release of the nascent chain bearing a 3′ puromycin (bottom). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Major applications of puromycin and its derivatives. (A) In SUrface SEnsing of Translation (SUnSET), global translation rates are estimated based on incorporation of puromycin into membrane proteins. After a pulse of puromycin in cultured cells or whole animals, puromycylated membrane proteins are trafficked to the plasma membrane, where they can be detected by FACS using anti-puromycin antibodies. (B) In Ribopuromycylation (RPM), cells are plated on cover slips and emetine is used to arrest elongating ribosomes. Subsequent addition of puromycin leads to incorporation into nascent chains without release from ribosomes. Cells are then fixed, permeabilized and stained with anti-puromycin antibodies to visualize translating ribosomes in situ. (C) In puro-PLA, puromycylation of cells on cover slips is followed by incubation with two primary antibodies: one against puromycin and another against a protein of interest. Oligonucleotide conjugated secondary antibodies are then added, and ligation takes place wherever the two primary antibodies bind closely to each other on the same protein molecule. After rolling circle amplification, the products are visualized by hybridization to a fluorescent oligonucleotide probe. This detects the subcellular localization of specific newly synthesized proteins. (D) In PUromycin-associated Nascent CHain Proteomics (PUNCH-P), translating ribosomes are extracted from cells or tissues and incubated with biotin-dC-puromycin. Puromycylated nascent chains are then isolated by streptavidin affinity purification and subjected to mass-spectrometry analysis, to generate a snapshot of the entire nascent proteome. (E) In mRNA display, a cDNA library is in vitro transcribed and an oligonucleotide spacer modified with a 3′ puromycin is covalently attached to each transcript. Using cell-free translation, incorporation of puromycin by ribosomes that reach the stop codon links the nascent polypeptide to its cognate transcript. These mRNA-protein fusion products are then selected by binding to bait proteins or nucleic acids and amplified by error-prone PCR for additional rounds of selection prior to detection by next generation sequencing.