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.