Simultaneous and functional binding of SmpB and EF-Tu center dot GTP to the alanyl acceptor arm of tmRNA

Transfer-messenger RNA (tmRNA) mimics functions of aminoacyl-tRNA and mRNA, subsequently, when rescuing stalled ribosomes on a 3' truncated mRNA witho ut stop codon in bacteria. In addition, this mechanism marks prematurely te rminated proteins by a C-terminal peptide tag as a signal for degradation b y specific cellular proteases. For Escherichia coli, previous studies on in itial steps of this "trans-translation" mechanism revealed that tmRNA alany lation by Ala-tRNA synthetase and binding of Ala-tmRNA by EF-Tu-GTP for sub sequent delivery to stalled ribosomes are inefficient when compared to anal ogous reactions with canonical tRNA(Ala). In other studies, protein SmpB an d ribosomal protein S1 appeared to bind directly to tmRNA and to be indispe nsable for trans-translation. Here, we have searched for additional and syn ergistic 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 efficiency 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 acceptor arm of Ala-tRNA(Ala), but there th e alanylation efficiency was inhibited rather than stimulated by SmpB. Ther efore, SmpB may function as an essential modulator of the tRNA-like accepto r arm of tmRNA during its successive steps in trans-translation. (C) 2001 A cademic Press.