Nucleic Acids Research Advance Access originally published online on April 17, 2009
Nucleic Acids Research 2009 37(11):3747-3755; doi:10.1093/nar/gkp240
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2009, Vol. 37, No. 11 3747-3755
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
RNA |
Low modularity of aminoacyl-tRNA substrates in polymerization by the ribosome
Department of Pharmacology and Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center, 2222 Pierce Ave., Nashville, TN 37232, USA
*To whom correspondence should be addressed. Tel: +1 615 936 3112; Fax: +1 615 936 5555; Email: a.forster{at}vanderbilt.edu
Received February 14, 2009. Accepted March 30, 2009.
Aminoacyl-transfer RNAs contain four standardized units: amino acids, an invariant 3'-terminal CCA, trinucleotide anticodons and tRNA bodies. The degree of interchangeability of the three variable modules is poorly understood, despite its role in evolution and the engineering of translation to incorporate unnatural amino acids. Here, a purified translation system is used to investigate effects of various module swaps on the efficiency of multiple ribosomal incorporations of unnatural aminoacyl-tRNA substrates per peptide product. The yields of products containing three to five adjacent L-amino acids with unnatural side chains are low and cannot be improved by optimization or explained simply by any single factor tested. Though combinations of modules that allow quantitative single unnatural incorporations are found readily, finding combinations that enable efficient synthesis of products containing multiple unnatural amino acids is challenging. This implies that assaying multiple, as opposed to single, incorporations per product is a more stringent assay of substrate activity. The unpredictability of most results illustrates the multifactorial nature of substrate recognition and the value of synthetic biology for testing our understanding of translation. Data indicate that the degree of interchangeability of the modules of aminoacyl-tRNAs is low.