title: The tRNA-like domains of E coli and A.aeolicus transfer-messenger RNA: structural and functional studies.
abstract: Transfer-messenger RNA (tmRNA, 10Sa RNA or ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. The understanding at molecular level of this ubiquitous quality control system in eubacteria requires structural information. Here, we describe the purification and structural analysis of a functional fragment of both Aquifex aeolicus and Escherichia coli tmRNA, recapitulating their tRNA-like domain, which were expressed in vivo from synthetic genes. Both recombinant RNA are correctly processed at both 5' and 3' ends and are produced in quantities suitable for structural analysis by NMR and/or X-ray crystallography. The sequence and solution structure of the tRNA-like domains were analysed by various methods including structural mapping with chemical and enzymatic probes and 2D NMR spectroscopy. The minimalist RNAs contain two post-transcriptional base modifications, 5-methyluridine and pseudouridine, as the full-length tmRNA. Both RNAs fold into three stems, a D-analogue, a T-loop and a GAAA tetra-loop. 2D NMR analysis of the imino proton resonances of both RNAs allowed the assignment of the three stems and of a number of tertiary interactions. It shows the existence of interactions between the TPsiC-loop and the D-analogue, exhibiting a number of similarities and also differences with the canonical tRNA fold, indicating that RNA tertiary interactions can be modulated according to the sequence and secondary structure contexts. Furthermore, the E.coli minimalist RNA is aminoacylatable with alanine with a catalytic efficiency an order of magnitude higher than that for full-length tmRNA.
authors.affiliation: Laboratoire de Biochimie Pharmaceutique UPRES JE2311, Faculté de Pharmacie, Université de Rennes I, 2 avenue du Pr. Léon Bernard, 35043, Rennes, France.
authors.forename: Cyril
authors.initials: C
authors.lastname: Gaudin
authors.forename: Sylvie
authors.initials: S
authors.lastname: Nonin-Lecomte
authors.forename: Carine
authors.initials: C
authors.lastname: Tisné
authors.forename: Sophie
authors.initials: S
authors.lastname: Corvaisier
authors.forename: Valérie
authors.initials: V
authors.lastname: Bordeau
authors.forename: Frédéric
authors.initials: F
authors.lastname: Dardel
authors.forename: Brice
authors.initials: B
authors.lastname: Felden
mesh_terms: D000409:Alanine / Q000737:chemistry; D001419:Bacteria / Q000378:metabolism*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012313:RNA; D012343:RNA, Transfer; D000409:Alanine