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Báo cáo khoa học: tmRNA from Thermus thermophilus Interaction with alanyl-tRNA synthetase and elongation factor Tu

The interaction of aThermus thermophilustmRNA tran-script with alanyl-tRNA synthetase and elongation factor Tu has been studied. The synthetic tmRNA was found to be stable up to 70 C. The thermal optimum of tmRNA alanylation was determined to be around 50 C. At 50 C, tmRNA transcript was aminoacylated by alanyl-tRNA synthetase with 5.9times lower efficiency (kcat /Km) than tRNA Ala , primarily because of the difference in turnover numbers (kcat ). Studies on EF-Tu protection of Ala tmRNA against alkaline hydrolysis revealed the existence of at least two different binding sites for EF-Tu on charged tmRNA. . | Eur. J. Biochem. 270 463-475 2003 FEBS 2003 doi 10.1046 j.1432-1033.2003.03401.x tmRNA from Thermus thermophilus Interaction with alanyl-tRNA synthetase and elongation factor Tu Victor G. Stepanov and Jens Nyborg Institute of Molecular and Structural Biology University of Aarhus Denmark The interaction of a Thermus thermophilus tmRNA transcript with alanyl-tRNA synthetase and elongation factor Tu has been studied. The synthetic tmRNA was found to be stable up to 70 C. The thermal optimum of tmRNA alanylation was determined to be around 50 C. At 50 C tmRNA transcript was aminoacylated by alanyl-tRNA synthetase with 5.9 times lower efficiency kcat Km than tRNAAla primarily because of the difference in turnover numbers kcat . Studies on EF-Tu protection of Ala tmRNA against alkaline hydrolysis revealed the existence of at least two different binding sites for EF-Tu on charged tmRNA. The possible nature of these binding sites is discussed. Keywords tmRNA elevated temperatures alanyl-tRNA synthetase EF-Tu. The transfer-messenger RNA tmRNA is a small stable bacterial RNA that is an object of considerable interest because of its obvious structural and functional dualism. This molecule possesses both mRNA and tRNA activities and contains easily recognizable mRNA-like and tRNA-like modules 1 . The latter is formed by converging-3 - and 5 -termini of the 300-400 nucleotide-long chain. The main biological function of tmRNA is to relieve ribosomes that remain for a long time in complex with mRNA without elongating the polypeptide chain. Such a situation arises upon translation of truncated mRNA deprived of stopcodon or intact mRNA with clustered rare codons. The intervention of tmRNA may also take place in the case when the ribosomes idle at the mRNA stop-codon awaiting proper termination of translation 2 . As a first step of the tmRNA-assisted ribosome rescue called trans-translation the aminoacylated tRNA-like module of the tmRNA binds to the A-site of stalled 70S ribosomes