Abstract
Transfer-messenger RNA (tmRNA) is a stable RNA in bacteria of 360 +/- 40 nucleotides that can be charged with alanine and can function as both tRNA and mRNA. Ribosomes that are stalled either in a coding region of mRNA or at the 3' end of an mRNA fragment lacking a stop codon are rescued by replacing their mRNA for tmRNA. Here we demonstrate that the interaction of tmRNA with the elongation factor Tu shows unexpected features. Deacylated tmRNA can form a complex with either EF-Tu.GDP or EF-Tu.GTP, the association constants are about one order of magnitude smaller than that of an Ala-tRNA.EF-Tu.GTP complex. tmRNA as well as Ala-tmRNA can be efficiently cross-linked with EF-Tu.GDP using a zero-length cross-link. The efficiency of cross-linking in the case of deacylated tmRNA does not depend on an intact CCA-3' end and is about the same, regardless whether protein mixtures such as the post-ribosomal supernatant (S100 enzymes) or purified EF-Tu are present. Two cross-linking sites with EF-Tu.GDP have been identified that are located outside the tRNA part of tmRNA, indicating an unusual interaction of tmRNA with EF-Tu.GDP.
Highlights
Transfer-messenger RNA is a stable RNA in bacteria of 360 ؎ 40 nucleotides that can be charged with alanine and can function as both tRNA and mRNA
The elongation factor elongation factor Tu (EF-Tu) within ternary complex aminoacyl-tRNA1⁄7EF-Tu1⁄7GTP increases the affinity of an aminoacyltRNA to the ribosomal A site by at least two orders of magnitude [34]
EF-Tu interacts with the short arm of an aa-tRNA that comprises the acceptor stem and the T loop-stem structure [35]
Summary
Transfer-messenger RNA (tmRNA) is a stable RNA in bacteria of 360 ؎ 40 nucleotides that can be charged with alanine and can function as both tRNA and mRNA. The main function of tmRNA in the cell seems to be the rescue of ribosomes that are arrested by truncated mRNA lacking their stop codon [8, 9]. These ribosomes carry a peptidyl-tRNA at the P site and either a truncated codon or no codon at all at the A site. After reading the codon sequence on tmRNA, the ribosome terminates at the stop codon encoded in tmRNA by the usual termination mechanism This process has two consequences: (i) the ribosomes arrested with truncated mRNA are rescued and recycled for protein synthesis, and (ii) the truncated proteins are tagged with a signal peptide that is recognized by specific proteases. It was demonstrated that stalling of a ribosome at rare codons can trigger tagging of polypeptides despite the presence of a substantial region of non-translated 3Ј-mRNA [11]
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