A hydantoin isoform of cyclic N6-threonylcarbamoyladenosine (ct6A) is present in tRNAs

Abstract

N6-Threonylcarbamoyladenosine (t6A) and its derivatives are universally conserved modified nucleosides found at position 37, 3΄ adjacent to the anticodon in tRNAs responsible for ANN codons. These modifications have pleiotropic functions of tRNAs in decoding and protein synthesis. In certain species of bacteria, fungi, plants and protists, t6A is further modified to the cyclic t6A (ct6A) via dehydration catalyzed by TcdA. This additional modification is involved in efficient decoding of tRNALys. Previous work indicated that the chemical structure of ct6A is a cyclic active ester with an oxazolone ring. In this study, we solved the crystal structure of chemically synthesized ct6A nucleoside. Unexpectedly, we found that the ct6A adopted a hydantoin isoform rather than an oxazolone isoform, and further showed that the hydantoin isoform of ct6A was actually present in Escherichia coli tRNAs. In addition, we observed that hydantoin ct6A is susceptible to epimerization under mild alkaline conditions, warning us to avoid conventional deacylation of tRNAs. A hallmark structural feature of this isoform is the twisted arrangement of the hydantoin and adenine rings. Functional roles of ct6A37 in tRNAs should be reconsidered.

Identification of 2-methylthio cyclic N6-threonylcarbamoyladenosine (ms2ct6A) as a novel RNA modification at position 37 of tRNAs

“…According to the structural analysis of ct 6 A nucleoside (15), two carbonyl oxygen atoms of the hydantoin ring are repulsive to the nitrogen atoms (N1 and N7) of the adenine base (Supplementary Data). Consistent with this, the C6-N6 bond length is longer than the normal C-N bond length.…”

“…Very recently, however, X-ray crystallography of synthetic ct 6 A nucleoside revealed the existence of a distinct isoform with a hydantoin structure (Supplementary Data) (15). LC/MS co-injection analyses showed that chemically synthesized ct 6 A nucleoside and natural ct 6 A in Escherichia coli tRNAs co-elute as a single peak by both reverse-phase and hydrophilic interaction liquid chromatography.…”

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

“…Commonly used protocols for digestion of tRNA involve sequential incubation with acidic and basic buffers for nuclease P1 and alkaline phosphatase, respectively. However, recent studies have reported that both acidic and basic reaction conditions lead to degradation of many labile modifications, such as ct 6 A to t 6 A and ms 2 ct 6 A to ms 2 t 6 A [59,87,88]. Alternatively, this problem can be overcome by digestion at physiological pH, and the use of endonucleases like benzonase, which is active at physiological pH [116].…”

“…Recently, X-ray crystallography, along with LC–MS analysis of native and synthetic ct 6 A enabled further characterization of the nucleoside in the hydantoin isoform, rather than the previously predicted oxazolone isoform (Figure 2), with a λ max of 269 nm. From the structure, it is not intuitive how the twisted position of the ring against the adenine base contributes to decoding efficiency and less understood are the effects of this further modification on translation [87,88]. …”

“…The proposed reaction mechanism likely involves the activation of the carboxyl group of the t 6 A threonyl moiety through adenylation. This step then enables the nucleophilic attack of the N6 nitrogen of t 6 A onto the activated carboxylate group, leading to formation of the hydantoin ring [87]. Interestingly, in E. coli the tcdA gene is located adjacent, yet in the opposite direction of csdA and csdE genes encoding a cysteine desulfurase and sulfur acceptor, respectively.…”