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

Published online by Cambridge University Press:  20 August 2002

SHELLEY R. STARCK
Affiliation:
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
RICHARD W. ROBERTS
Affiliation:
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
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Abstract

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.

Type
Research Article
Copyright
2002 RNA Society

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