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J. Biol. Chem., Vol. 281, Issue 45, 34258-34268, November 10, 2006

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Prolyl-tRNA^Pro in the A-site of SecM-arrested Ribosomes Inhibits the Recruitment of Transfer-messenger RNA^*

From the Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106

Translational pausing can lead to cleavage of the A-site codon and facilitate recruitment of the transfer-messenger RNA (tmRNA) (SsrA) quality control system to distressed ribosomes. We asked whether aminoacyl-tRNA binding site (A-site) mRNA cleavage occurs during regulatory translational pausing using the Escherichia coli SecM-mediated ribosome arrest as a model. We find that SecM ribosome arrest does not elicit efficient A-site cleavage, but instead allows degradation of downstream mRNA to the 3'-edge of the arrested ribosome. Characterization of SecM-arrested ribosomes shows the nascent peptide is covalently linked via glycine 165 to in the peptidyl-tRNA binding site, and is bound to the A-site. Although A-site-cleaved mRNAs were not detected, tmRNA-mediated ssrA tagging after SecM glycine 165 was observed. This tmRNA activity results from sequestration of on overexpressed SecM-arrested ribosomes, which produces a second population of stalled ribosomes with unoccupied A-sites. Indeed, compensatory overexpression of readily inhibits ssrA tagging after glycine 165, but has no effect on the duration of SecM ribosome arrest. We conclude that, under physiological conditions, the architecture of SecM-arrested ribosomes allows regulated translational pausing without interference from A-site cleavage or tmRNA activities. Moreover, it seems likely that A-site mRNA cleavage is generally avoided or inhibited during regulated ribosome pauses.

Received for publication, August 22, 2006 , and in revised form, September 7, 2006.

^* This work was supported by start-up funds from the University of California, Santa Barbara, and grants from Santa Barbara Cottage Hospital and the University of California, Santa Barbara. 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 Both authors contributed equally to the results of this study.

² To whom correspondence should be addressed: Life Sciences Technology Bldg., Rm. 3105, University of California, Santa Barbara, CA 93106-9610. Tel.: 805-893-2028; Fax: 805-893-4724; E-mail: chayes{at}lifesci.ucsb.edu.

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