Tertiary structure of animal tRNA^Trp in solution and interaction of tRNA^Trp with tryptophanyl‐tRNA synthetase

Abstract

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N-7 in guanosines and N-3 in cytidines by dimethyl sulfate and carbethoxylation of N-7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNAT'p in solution as well as its interactions with tryptophanyl-tRNA synthetase.The phosphate reactivity towards ethylnitrosoureaof unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and TY arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPh" since beef tRNATrp showed a more stable D stem and a less stable T Y stem.When alkylation by ethylnitrosourea was studied with the tRNATrp. tryptophanyl-tRNA synthetase complex we found that phosphates located at the 5' side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N-3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase . tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl-tRNA synthetase was confirmed in nuclease S1 mapping experiments.After a quarter of a century of intensive work, the role of tRNA in protein biosynthesis has been well established [l]. However this is not the only cellular role played by these molecules. Transfer RNAs are also involved in processes as different as the biosynthesis of the bacterial cell wall, the regulation of transcription through the mechanism of attenuation and the expression of retroviruses [2, 31.The primary structure of several hundred tRNAs is known [4], while the tridimensional structure of only two elongator tRNAs is known at high resolution [5-91. These structures have been determined by X-ray diffraction of tRNA crystals. However, only some tRNAs are able to give crystals suitable for X-ray diffraction studies, so that other techniques for studying tRNA structure in solution have been developed. In general good agreement is obtained between results of diffraction and solution studies [lo-121. One of these techniques for the study of tRNA in solution consists in alkylating unspecifically the phosphate groups of the tRNA backbone with the reagent N-ethylnitrosourea. This substance will modify only those phosphate groups not shielded by the three-dimensional structure of the molecule [lo]. Other reagents used in recent years for the modification of nucleic acids are dimethyl sulfate which alkylates the N-3 position of cytidines and ...

An unusual RNA tertiary interaction has a role for the specific aminoacylation of a transfer RNA.

“…The nucleotides at position 48 in other tRNAs were also protected from chemical probes (17). However, the reactivity ofG15 is in contrast to what has been shown for E. coli tRNAThr (17), beef tRNATrP (26), yeast tRNAPhe (27), and tRNAASP (27), where purine-15 in a normal Levitt pair is stacked on A14 and is not reactive. Additional model-building suggests an alternative base-pairing scheme between G15 and G48 that maintains the trans base pairing of the sugar phosphate backbone but forms two hydrogen bonds using N-2 hydrogen as the donor and ring N-3 as the acceptor (Fig.…”

Tertiary structure of Escherichia coli tRNAThr3 in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

“…Of interest is the fact that mapping of the complexed tRNA was done under similar pH conditions (pH 7.5 -8.0) with all structural probes. This was in particular the case for nuclease S1 which shows an optimal reactivity in the acidic range, at pH 4.5 [47] but can be used at pH 7.5 [17,191. Such conditions, with pH above 7.0, are known to be favourable for specific tRNA/synthetase interactions [41].…”

tRNATrp as cofactor of gelonin, a ribosome‐inactivating protein with RNA‐N‐glycosidase activity features required for the cofactor activity

“…The fact that only the mammalian tRNATrp is active in promoting inactivation of ribosomes by gelonin indicates that the identity elements involved in this activity are different from those recognised by the synthetase. The two tgNAs share the discriminator base and most of the anticodon loop, which are considered the identity elements for tryptophanyl-tRNA synthetase (5,20), but their St~luences differ by 24 bases out of 75 (20). These differences should provide the basis for the Artemia salina ribosomes (10 pmol) were preincubated with 5 nM gelonin, a concentration of the RIP well below that required for the inactivation of ribosomes in the absence of cofactors (1,17), for 10 rain at 28°C in 10 Ixl of 10 mM Tris-HCl, pH 7.4, containing 100 mM ammonium acetate, 2 mM magnesium acetate and different amounts (5-300 ng) of each tKNA.…”