Simultaneous and Functional Binding of SmpB and EF-Tu  GTP to the Alanyl Acceptor Arm of tmRNA Sharief Barends 1 , A. Wali Karzai 2 , Robert T. Sauer 2 , Jacek Wower 3 and Barend Kraal 1 * 1 Department of Biochemistry Leiden Institute of Chemistry Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands 2 Department of Biology Massachusetts Institute of Technology, Cambridge MA 02139, USA 3 Department of Animal and Dairy Sciences, Program in Cell and Molecular Biosciences Auburn University, 210 Upchurch Hall, Auburn AL 36849-5415, USA Transfer-messenger RNA (tmRNA) mimics functions of aminoacyl-tRNA and mRNA, subsequently, when rescuing stalled ribosomes on a 3 0 trun- cated mRNA without stop codon in bacteria. In addition, this mechanism marks prematurely terminated proteins by a C-terminal peptide tag as a signal for degradation by speci®c cellular proteases. For Escherichia coli, previous studies on initial steps of this ``trans-translation'' mechanism revealed that tmRNA alanylation by Ala-tRNA synthetase and binding of Ala-tmRNA by EF-Tu  GTP for subsequent delivery to stalled ribo- somes are inef®cient when compared to analogous reactions with canoni- cal tRNA Ala . In other studies, protein SmpB and ribosomal protein S1 appeared to bind directly to tmRNA and to be indispensable for trans- translation. Here, we have searched for additional and synergistic effects of the latter two on tmRNA alanylation and its subsequent binding to EF-Tu  GTP. Kinetic analysis of functioning combined with band-shift experiments and structural probing demonstrate, that tmRNA may indeed form a multimeric complex with SmpB, S1 and EF-Tu  GTP, which leads to a considerably enhanced ef®ciency of the initial steps of trans-translation. Whereas S1 binds to the mRNA region of tmRNA, we have found that SmpB and EF-Tu both interact with its acceptor arm region. Interaction with SmpB and EF-Tu was also observed at the accep- tor arm of Ala-tRNA Ala , but there the alanylation ef®ciency was inhibited rather than stimulated by SmpB. Therefore, SmpB may function as an essential modulator of the tRNA-like acceptor arm of tmRNA during its successive steps in trans-translation. # 2001 Academic Press Keywords: Ala-tRNA synthetase; mutant tmRNA; ribosomal protein S1; trans-translation; tRNA Ala *Corresponding author Introduction Transfer-messenger RNA (tmRNA) was ®rst identi®ed in Escherichia coli as the rescue molecule mediating a so-called trans-translation mechan- ism 1 ± 4 and appears to occur in all eubacteria. 5,6 The mechanism is activated when ribosomes are stalled either at the 3 0 end of a truncated messenger RNA without an in-frame stop codon or at clusters of rare codons in mRNA. 7 Alanyl-tmRNA (Figure 1) enters the ribosome at the non-pro- grammed and/or empty A-site and donates its ala- nine residue to the nascent peptide chain. Thereafter, the ribosome hops from the dead-end of the mRNA to a short reading frame in the tmRNA and is ®nally released by termination at a proper stop codon. The truncated protein product is thus fused to a C-terminal peptide tag encoded by tmRNA, which makes it a substrate for protein degradation (for review and databases see Karzai et al., 4 Williams 8 and Zwieb & Wower 9 ). The mech- anism functions for the removal of truncated and unwanted protein intermediates, and can regulate speci®c gene expression. For instance, in the case of coupled transcription-translation of the lac repressor gene, tmRNA action plays a role in the regulation of active repressor levels. 10 Present address: A. W. Karzai, 410 Life Science Building, Department of Biochemistry and Cell Biology, SUNY at Stony Brook, NY 11790, USA. Abbreviations used: AlaRS, Ala-tRNA synthetase; PEP, phosphoenolpyruvate; PK, pyruvate kinase; tmRNA, transfer-messenger RNA. E-mail address of the corresponding author: b.kraal@chem.leidenuniv.nl doi:10.1006/jmbi.2001.5114 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 314, 9±21 0022-2836/01/010009±13 $35.00/0 # 2001 Academic Press