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J. Biol. Chem., Vol. 282, Issue 48, 34779-34786, November 30, 2007

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Functional SmpB-Ribosome Interactions Require tmRNA^*

Thomas R. Sundermeier and A. Wali Karzai¹

From the Department of Biochemistry and Cell Biology and the Center for Infectious Diseases, Stony Brook University, Stony Brook, New York 11794

Small protein B (SmpB) is a requisite component of the transfer messenger RNA (tmRNA)-mediated bacterial translational quality control system known as trans-translation. The initial binding of tmRNA and its subsequent accommodation into the ribosomal A-site are activities intimately linked to SmpB protein function. From a mechanistic perspective, two key unanswered questions that require further investigation are: 1) what constitutes a stalled ribosome recognition complex and 2) does SmpB pre-bind ribosomes to recruit tmRNA. We have assessed, both in vivo and in vitro, the nature and stability of free SmpB interactions with stalled ribosomes and examined whether these interactions are functionally relevant. We present evidence to demonstrate that interaction of free SmpB with ribosomes is salt sensitive and significantly more labile than interaction of the SmpB·tmRNA complex with ribosomes. Upon dissociation of 70 S ribosomes SmpB partitions primarily with tmRNA rather than ribosomal subunits. This finding is consistent with biochemical and structural data demonstrating that tmRNA is the high-affinity binding partner of SmpB. Moreover, we show that under normal physiological conditions roughly similar numbers of SmpB and tmRNA molecules are present in cells. Our investigations also reveal that upon induction of a nonstop mRNA, SmpB is enriched in stalled ribosome fractions only in the presence of tmRNA. Based on these findings, we conclude that SmpB does not pre-bind stalled ribosome and that functional SmpB-stalled ribosome interactions require tmRNA. We propose that a 1:1:1 complex of SmpB·tmRNA·EF-Tu(GTP) recognizes and binds a stalled ribosome to initiate trans-translation.

Received for publication, August 29, 2007 , and in revised form, October 2, 2007.

^* This work was supported in part by National Institutes of Health Grants GM65319 and AI055621 (to A. W. K.), The Northeast Biodefense Center, and The Pew Scholars Program. 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 To whom correspondence should be addressed. Tel.: 631-632-1688; Fax: 631-632-8575; E-mail: akarzai{at}ms.cc.sunysb.edu.

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