Starck, Shelley R. and Roberts, Richard W. (2002) Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition. RNA, 8 (7). pp. 890-893. ISSN 1355-8382. http://resolver.caltech.edu/CaltechAUTHORS:20111102-135333071
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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 (IC_50 < 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 tRNA^(Ala)-derived minihelices containing puromycin provide an exception to the size trend, and are the only constructs longer than 4 nt with any appreciable potency (IC_50 = 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.
|Additional Information:||© 2002 RNA Society. Received February 22, 2002; returned for revision March 23, 2002; revised manuscript received April 19, 2002. We thank Jie Xu for synthesis of N-trifluoroacetyl-puromycin, and Terry T. Takahashi for comments on the manuscript. This work was supported by National Institutes of Health (NIH) Grant R01 GM60416 to R.W.R. and by NIH training grant GM 07616 (S.R.S.).|
|Subject Keywords:||eukaryotes; in vitro translation; mRNA display; protein synthesis inhibitors; reticulocyte lysate; ribosomes|
|Official Citation:||SHELLEY R. STARCK and RICHARD W. ROBERTS (2002). Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition. RNA, 8 , pp 890-903 doi:10.1017.S1355838202022069|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Jason Perez|
|Deposited On:||03 Nov 2011 21:57|
|Last Modified:||03 Nov 2011 21:57|
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