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Nucleic Acids Research Advance Access published online on November 12, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp1025
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© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Quantitative analysis of ribosome–mRNA complexes at different translation stages

Nikolay E. Shirokikh1, Elena Z. Alkalaeva2, Konstantin S. Vassilenko1, Zhanna A. Afonina1, Olga M. Alekhina1, Lev L. Kisselev2 and Alexander S. Spirin1,*

1Laboratory of Mechanisms of Protein Biosynthesis, Institute of Protein Research, Russian Academy of Sciences, 4 Institutskaya St., Pushchino, Moscow Region, 142290 and 2Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova St., Moscow, 119991, Russia

*To whom correspondence should be addressed. Tel: +7 4967 73 06 69; Fax: +7 499 632 78 71; Email: spirin{at}vega.protres.ru

Received September 24, 2009. Revised October 18, 2009. Accepted October 20, 2009.

Inhibition of primer extension by ribosome–mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture.


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