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J. Biol. Chem., Vol. 283, Issue 26, 18431-18440, June 27, 2008

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Perturbation of the tRNA Tertiary Core Differentially Affects Specific Steps of the Elongation Cycle^*

Dongli Pan, Chun-Mei Zhang¹, Stanislav Kirillov², Ya-Ming Hou, and Barry S. Cooperman³

From the Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323 and the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

The tRNA tertiary core region is important for both tRNA stability and activity in the translation elongation cycle. Here we report the effects of mutating each of two highly conserved base pairs in the tertiary core of Phe-tRNA^Phe, 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of aminoacyl-tRNA·EF-Tu·GTP ternary complexs and culminating with translocation of A-site-bound peptidyl-tRNA into the P-site. We find that codon-dependent binding of aminoacyl-tRNA to the A/T-site and proofreading of near-cognate tRNA are sensitive to perturbation of either base pair; formation of the ternary complex and accommodation from the A/T to the A-site are sensitive to 18-55 perturbation only, and translocation of peptidyl-tRNA from the A- to P-site is insensitive to perturbation of either. These results underline the importance of the core region in promoting the efficiency and accuracy of translation, and they likely reflect different requirements for structural integrity of the core during specific steps of the elongation cycle.

Received for publication, February 26, 2008 , and in revised form, April 30, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM071014 (to B. S. C.) and GM56662 (to Y.-M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Vector BioLabs, Inc., 3701 Market St., Philadelphia, PA 19104.

² Present address: Petersburg Nuclear Physics Institute RAS, 188300 Gatchina, Russia.

³ To whom correspondence should be addressed. Tel.: 215-898-6330; Fax: 215-898-2037; E-mail: cooprman{at}pobox.upenn.edu.

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