Rapid selection of aminoacyl-tRNAs based on biotinylation of -NH2 group of charged amino acids

International audienceA rapid selection procedure to separate low amounts of aminoacylated tRNAs from large pools of inactive variants is described. The procedure involves a three-step protocol. After initial aminoacylation of a tRNA pool, N-hydroxysuccinimide ester chemistry is applied to biotinylate the alpha-NH2 group of the amino acid bound to the 3'-end of a tRNA. The biotin tag is used to capture the derivatized tRNAs on streptavidin-conjugated magnetic beads. Variants bound to the solid phase can be amplified by RT-PCR and transcription, providing tRNAs for subsequent selection rounds

Selection of viral RNA-derived tRNA-like structures with improved valylation activities.

The tRNA-like structure (TLS) of turnip yellow mosaic virus (TYMV) RNA was previously shown to be efficiently charged by yeast valyl-tRNA synthetase (ValRS). This RNA has a noncanonical structure at its 3'-terminus but mimics a tRNA L-shaped fold, including an anticodon loop containing the major identity nucleotides for valylation, and a pseudoknotted amino acid accepting domain. Here we describe an in vitro selection experiment aimed (i) to verify the completeness of the valine identity set, (ii) to elucidate the impact of the pseudoknot on valylation, and (iii) to investigate whether functional communication exists between the two distal anticodon and amino acid accepting domains. Valylatable variants were selected from a pool of 2 x 10(13) RNA molecules derived from the TYMV TLS randomized in the anticodon loop nucleotides and in the length (1-6 nucleotides) and sequence of the pseudoknot loop L1. After nine rounds of selection by aminoacylation, 42 have been isolated. Among them, 17 RNAs could be efficiently charged by yeast ValRS. Their sequence revealed strong conservation of the second and the third anticodon triplet positions (A(56), C(55)) and the very 3'-end loop nucleotide C(53). A large variability of the other nucleotides of the loop was observed and no wild-type sequence was recovered. The selected molecules presented pseudoknot domains with loop L1 varying in size from 3-6 nucleotides and some sequence conservation, but did neither reveal the wild-type combination. All selected variants are 5-50 times more efficiently valylated than the wild-type TLS, suggesting that the natural viral sequence has emerged from a combination of evolutionary pressures among which aminoacylation was not predominant. This is in line with the role of the TLS in viral replication.journal articleresearch support, non-u.s. gov't2000 May 23importe

Rapid selection of aminoacyl-tRNAs based on biotinylation of alpha-NH2 group of charged amino acids.

A rapid selection procedure to separate low amounts of aminoacylated tRNAs from large pools of inactive variants is described. The procedure involves a three-step protocol. After initial aminoacylation of a tRNA pool, N-hydroxysuccinimide ester chemistry is applied to biotinylate the alpha-NH2 group of the amino acid bound to the 3'-end of a tRNA. The biotin tag is used to capture the derivatized tRNAs on streptavidin-conjugated magnetic beads. Variants bound to the solid phase can be amplified by RT-PCR and transcription, providing tRNAs for subsequent selection rounds.journal articleresearch support, non-u.s. gov't1997 May 01importe

ED-TRA - Engineering Desktop Anwendungen in der Tragwerksplanung Schlussbericht

Available from TIB Hannover: F02B231 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Bildung und Forschung, Berlin (Germany)DEGerman

Ribosome-induced RNA conformational changes in a viral 3′-UTR sense and regulate translation levels

Complex RNA three-dimensional structures undergo functionally important programmed conformational changes. Here, the authors report how two structurally and functionally coupled RNA domains within a viral 3′-UTR sense the ribosome through conformational changes and respond by modulating translation