Aminoacylated tmRNA from Escherichia coli interacts with prokaryotic elongation factor Tu.

Eubacterial tmRNAs (10Sa RNAs) are unique because they function, at least in Escherichia coli, both as tRNA and mRNA (for a review, see Muto et al+, 1998)+ These ;360 6 40-nt-long RNAs are charged with alanine at their 39 ends by alanyl-tRNA synthetases or AlaRS (Komine et al+, 1994; Ushida et al+, 1994)+ Alanylation occurs thanks to the presence of the equivalent of the G3-U70 pair, the major identity element for the alanylation of canonical tRNAs (Hou & Schimmel, 1988; McClain & Foss, 1988)+ Bacterial tmRNAs also have a short reading frame coding for 9 to 27 amino acids, depending on the species+ E. coli tmRNA mediates recycling of ribosomes stalled at the end of terminatorless mRNAs, via a trans-translation process (Tu et al+, 1995; Keiler et al+, 1996; Himeno et al+, 1997;Withey & Friedman, 1999)+ In E. coli, this amino acid tag is cotranslationally added to polypeptides synthesized from mRNAs lacking a termination codon, and the added 11-amino-acid C-terminal tag makes the protein a target for specific proteolysis (Keiler et al+, 1996)+ Structural analyses based on phylogenetic (Felden et al+, 1996; Williams & Bartel, 1996) and probing (Felden et al+, 1997; Hickerson et al+, 1998) data have led to a compact secondary structure model of E. coli tmRNA+ These molecules have structural similarities with canonical tRNAs, especially with tRNA acceptor branches (Fig+ 1)+ Further E. coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine, located in the T-loop mimic of the molecule (Felden et al+, 1998)+ Its mimicry with tRNA occurs also at the functional level, as it was already shown that tmRNA interacts with certain tRNA specific proteins such as RNase P (Komine et al+, 1994), RNase III (Srivastava et al+, 1990; Makarov & Apirion, 1992), AlaRS, the alanyl-tRNA synthetase (Komine et al+, 1994; Ushida et al+, 1994), and tRNA modifying enzymes (Felden, unpubl+ results)+ What about the other tRNA specific proteins involved in translation? E. coli tmRNA is found associated with 70S ribosomes in vivo, at about one molecule per 10 ribosomes (Ushida et al+, 1994; Komine et al+, 1996)+ How tmRNA enters the ribosomal A-site remains unknown+ It could either form a ternary complex after alanylation with elongation factor Tu (EF-TU) and guanosine-59-triphosphate (GTP), or use another specific pathway+ Biochemical studies (Rudinger et al+, 1994) as well as the X-ray structures of two ternary complexes (Nissen et al+, 1995, 1999) indicate that tRNAs interact with prokaryotic EFTu-GTP via the aminoacylated 39 end, the phosphorylated 59 end, and the first 10 bp of the acceptor branch+ Crystal structures also show that EF-Tu contacts mainly the ribose–phosphate backbone of the acceptor branch, thus accommodating sequence variability within elongator tRNAs+ E. coli tmRNA contains a 7-bp acceptor stem followed by a 5-bp T-stem, a 7-nt T-loop, and a classical XCCA single-stranded 39 end (Fig+ 1B), as in canonical elongator tRNAs+ A sequence comparison between E. coli tmRNA and tRNAAla acceptor branches indicates that 6 out of the first 10 bp of tmRNA are identical to that of tRNAAla (Fig+ 1)+ This suggests that alanylated tmRNA could be recognized by activated EF-Tu+ The aim of this study was to test the above assumption and to discover whether aminoacylated tmRNA forms a ternary complex with Thermus thermophilus EF-Tu and GTP+ The choice of the thermophilic protein was dictated by the available crystal structures of ternary complexes comprising this factor, considered to be canonical models for any ternary complex (Nissen et al+, 1995, 1999)+ Hydrolysis-protection assays were used to monitor the ternary complex formation+ They are based on the fact that the activated factor, when Reprint requests to: Brice Felden, Department of Human Genetics, University of Utah, 15N 2030E Room 6250, Salt Lake City, Utah 84112-5330, USA; e-mail: bfelden@genetics+utah+edu+ RNA (1999), 5:989–992+ Cambridge University Press+ Printed in the USA+ Copyright © 1999 RNA Society+