tRNA elbow modifications affect the tRNA pseudouridine synthase TruB and the methyltransferase TrmA

Abstract

tRNAs constitute the most highly modified class of RNA. Every tRNA contains a unique set of modifications, and Ψ55, m 5 U54, and m 7 G46 are frequently found within the elbow of the tRNA structure. Despite the abundance of tRNA modifications, we are only beginning to understand the orchestration of modification enzymes during tRNA maturation. Here, we investigated whether pre-existing modifications impact the binding affinity or catalysis by tRNA elbow modification enzymes. Specifically, we focused on the Escherichia coli enzymes TruB, TrmA, and TrmB which generate Ψ55, m 5 U54, and m 7 G46, respectively. tRNAs containing a single modification were prepared, and the binding and activity preferences of purified E. coli TrmA, TruB, and TrmB were examined in vitro. TruB preferentially binds and modifies unmodified tRNA. TrmA prefers to modify unmodified tRNA, but binds most tightly to tRNA that already contains Ψ55. In contrast, binding and modification by TrmB is insensitive to the tRNA modification status. Our results suggest that TrmA and TruB are likely to act on mostly unmodified tRNA precursors during the early stages of tRNA maturation whereas TrmB presumably acts on later tRNA intermediates that are already partially modified. In conclusion, we uncover the mechanistic basis for the preferred modification order in the E. coli tRNA elbow region.

Post-Transcriptional Modifications of Conserved Nucleotides in the T-Loop of tRNA: A Tale of Functional Convergent Evolution

“…Since the presence of N 1 -methyl group on A58 is required for efficient enzymatic transfer of sulfur on the C 2 -atom of m 5 U54 [ 113 ], the biogenesis of m 5 s 2 U54 by TtuA/TtuB/TtuC at elevated temperature in thermophilic Bacteria and possibly of m 1 s 4 Ψ in hyperthermophilic Archaea should occur later during tRNA maturation. These observations are in complete agreement with the theory of interdependency between the various enzymatic modification systems acting on nucleotides in the tRNA elbow, in which Ψ55-synthase TruB/PUS4/TRUB1 (and probably Pus10) appears to be the conductor, besides acting itself as a chaperone protein [ 136 , 137 , 138 , 139 ]. Interdependency between different nucleotide modifications and their modification pathways has been found to be particularly important for modifications in the anticodon-stem loop [ 60 , 140 , 141 ].…”

RNA nucleotide methylation: 2021 update

“…A specific mapping method found m 5 U outside tRNA and rRNA in eukaryotes (Carter et al, 2019). tRNA methylation by bacterial TrmA is efficient on unmodified tRNA transcript, suggesting this methylation to be an early event of tRNA maturation (Schultz & Kothe, 2020).…”

Molecular mechanism of tRNA binding by theEscherichia coliN7 guanosine methyltransferase TrmB

“…As we will show below, this variant is deficient only in methylation, but not tRNA binding. In the presence of SAM, wildtype TrmB forms product (26,29). Thus, the fluorescence decrease reflects an event related to tRNA binding prior to methylation.…”

“…Plasmids encoding wildtype or variant TrmB were transformed into BL21 (DE3) cells. Similarly, plasmids pBH113 and pBH402 for ThiI and IscS overexpression were transformed into BL21 (DE3) for overexpression as described in (29). In brief, for overexpression, cells were grown at 37°C in LB broth supplemented with 50 µg/mL kanamycin or 100 µg/mL ampicillin.…”