Skip to main content
    • Aa
    • Aa

Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition


Peptidyl transferase inhibitors have generally been studied using simple systems and remain largely unexamined in in vitro translation extracts. Here, we investigate the potency, product distribution, and mechanism of various puromycin–oligonucleotide conjugates (1 to 44 nt with 3′-puromycin) in a reticulocyte lysate cell-free translation system. Surprisingly, the potency decreases as the chain length of the oligonucleotide is increased in this series, and only very short puromycin conjugates function efficiently (IC50 < 50 μM). This observation stands in contrast with work on isolated large ribosomal subunits, which indicates that many of the puromycin–oligonucleotide conjugates we studied should have higher affinity for the peptidyl transferase center than puromycin itself. Two tRNAAla-derived minihelices containing puromycin provide an exception to the size trend, and are the only constructs longer than 4 nt with any appreciable potency (IC50 = 40–56 μM). However, the puromycin minihelices inhibit translation by sequestering one or more soluble translation factors, and do not appear to participate in detectable peptide bond formation with the nascent chain. In contrast, puromycin and other short derivatives act in a factor-independent fashion at the peptidyl transferase center and readily become conjugated to the nascent protein chain. However, even for the short derivatives, much of the translation inhibition occurs without peptide bond formation between puromycin and the nascent chain, a revision of the classical model for puromycin function. This peptide bond-independent mode is likely a combination of multiple effects including inhibition of initiation and failure to properly recycle translation complexes that have reacted with puromycin.

Corresponding author
Reprint requests to: Richard W. Roberts, Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA; e-mail:
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

  • ISSN: 1355-8382
  • EISSN: 1469-9001
  • URL: /core/journals/rna
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Full text views

Total number of HTML views: 0
Total number of PDF views: 2 *
Loading metrics...

Abstract views

Total abstract views: 167 *
Loading metrics...

* Views captured on Cambridge Core between September 2016 - 24th October 2017. This data will be updated every 24 hours.