The human tRNA-guanine transglycosylase displays promiscuous nucleobase preference but strict tRNA specificity

Abstract

Base-modification can occur throughout a transfer RNA molecule; however, elaboration is particularly prevalent at position 34 of the anticodon loop (the wobble position), where it functions to influence protein translation. Previously, we demonstrated that the queuosine modification at position 34 can be substituted with an artificial analogue via the queuine tRNA ribosyltransferase enzyme to induce disease recovery in an animal model of multiple sclerosis. Here, we demonstrate that the human enzyme can recognize a very broad range of artificial 7-deazaguanine derivatives for transfer RNA incorporation. By contrast, the enzyme displays strict specificity for transfer RNA species decoding the dual synonymous NAU/C codons, determined using a novel enzyme-RNA capture-release method. Our data highlight the broad scope and therapeutic potential of exploiting the queuosine incorporation pathway to intentionally engineer chemical diversity into the transfer RNA anticodon.

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit

“…In contrast, in WT cells only guanosines that were not replaced by Q despite the presence of a functional enzyme remained for the in vitro reaction. Unlike observed for the bTGT, the hTGT incorporated the preQ 1 -ligands to differing degrees, indicating a higher ability to distinguish between these analogues in accordance with previously published results by Kelly and co-workers ( 41 ). Additionally, incubation of in vitro transcribed tRNAs Asp, His, Tyr and Asn with human TGT and preQ 1 -L1 showed successful incorporation of the analogue into all of the four tRNAs ( Supplementary Figure S4a ).…”

“…via an incorporation of q-derivatives through transglycosylation. Apart from their natural substrates, both bTGT and eTGT have been shown to tolerate a certain variety of synthetic analogues harbouring large functional groups in vitro ( 41 , 42 ). Leveraging the short hairpin recognition motif of the bTGT installed on different RNA transcripts, Devaraj and co-workers developed a method called RNA-TAG ( t ransglycosylation a t g uanosine), allowing to site-specifically incorporate analogues in vitro , which contained large fluorophores or affinity labels for pull-down experiments.…”

Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase

“…Moreover, the molecular masses of QTRT1 and QTRT2 (variants) were measured to verify identity, integrity, and homogeneity, each. They were in excellent agreement with the respective calculated molecular masses [calculated/measured: QTRT1: 44 For the dual expression of the QTRT1 gene and the QTRT2 gene, both codon-optimized genes 19 were (separately from each other) PCR-amplified with the codons encoding the N-terminal 10 amino acids of QTRT1 (UniProtKB: Q9JMA2) and the codon encoding the N-terminal methionine of QTRT2 (UniProtKB: B8ZXI1) being omitted. Via the primers used for amplification, a Strep-tag II encoding sequence followed by a sequence encoding a PreScission Protease cleavage site were fused in frame to the 5′ end of the QTRT2 gene.…”

“…Clearly, in this conformation, it is unable to stabilize a nearby negative charge within the active center. For the same reasons, the insertion of further 7-(aminomethyl)-7-deazaguanine derivatives like the preQ 1 base, dihydroqueuine, or NPPDAG into tRNA occurs irreversibly. A further nucleobase, whose TGT-catalyzed insertion into tRNA was reported to be irreversible, is 7-deazaguanine .…”

“…In contrast, the TGT-catalyzed insertion of 8-azaguanine and of the preQ 0 base into tRNA is fully reversible. ,, While 8-azaguanine-34 is excised from the tRNA via the same mechanism as guanine-34, the 7-substituent of preQ 0 -34 does not allow the binding of a water molecule, which might serve as a donor for re-protonation and, thus, as the final acceptor of the negative charge. However, the 7-nitrile substituent of preQ 0 is conjugated with the 7-deazapurine scaffold.…”