The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs

Abstract

We have constructed eight anticodon-modified Escherichia coli initiator methionine (Met) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: (a) the stability of these eubacterial tRNA variants in the eukaryotic oocytes; (b) the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and (c) aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases.In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-~-~-ribofuranosylpurine-6-yl)carbamoy~]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.Some time ago a correlation between the anticodon sequence of a tRNA and the identity of neighbouring modified nucleosides was noted [l] (reviewed in [2]). Thus, the hypermodified nucleoside t6A, N-[N-(9-P-~-ribofuranosylpurine-6-yl)carbamoyl]threonine, or a derivative thereof is located in position 37 of tRNAs having anticodons terminating in a uridine. One enigmatic exception is the initiator methionine tRNA of Escherichia coli, for most other tRNAs including the eubacterial elongator Met-tRNA, which has the same anticodon sequence, and the eukaryotic initiator tRNA contain the A-37 modification [3].To study the effect of structural modifications in the anticodon loop on the modification of A-37, we have turned to recombinant RNA methods based on T4 RNA ligase. These techniques are particularly well-suited to the preparation of related tRNA chimera, which have substitutions in or near the anticodon, since fragments serving as starting material for the tRNA variant can be readily obtained from controlled

The synthesis and functional evaluation of RNA and DNA polymers having the sequence of Escherichia coli tRNAfMet

“…Threonylation experiments of ligRNA and rA77-tDNA were made possible by the finding that a yeast extract can recognize E. coli tRNAfMe' as a threonylation substrate much as in the case of frog oocytes [19]. Although IigRNA shows only modest activity, this activity was fully reproducible, could be verified by alternative analytical methodologies and is similar to the level of in vitro modification of an in vitro transcribed RNA [23].…”

“…In yeast, the adenosine 3' adjacent to the anticodon of initiator tRNAMe', A37, is modified with a threonyl substituent. Although the E. coli counterpart, tRNAfMc' does not contain this modification in vivo, threonylation of its A3' can be accomplished by injection into frog oocytes [19]. Here, we attempted to use another eukaryotic source, a yeast extract to threonylate the synthetic RNA.…”

Diversity of the biosynthesis pathway for threonylcarbamoyladenosine (t6A), a universal modification of tRNA

“…12 The hypermodified base t 6 A is present in nearly all ANN decoding tRNAs and has been studied in vitro and in vivo for more than 40 y. [13][14][15][16][17][18][19] Since the first discovery of the modification by Schweizer, et al in 1969, 18 sporadic studies established the basic requirements for the synthesis of this universal modification, identifying the requirement for ATP, threonine and carbonate, 15,17,[20][21][22] but fell short of elucidating the multi-step path to its formation. Subsequent studies in which native E. coli tRNA fMet (harboring an unmodified A 37 ) and yeast tRNA iMet transcripts were converted to t 6 A 37 after microinjection into Xenopus laevis oocytes demonstrated that the formation of t 6 A occurred in the oocyte cytoplasm and used a conserved machinery.…”

Cross Kingdom Functional Conservation of the Core Universally Conserved Threonylcarbamoyladenosine tRNA Synthesis Enzymes

“…N6-threonyl-carbamoyl-adenosine (t 6 A), a modification of adenosine located at position 37 next to the anticodon (t 6 A 37 ), is a highly conserved tRNA modification present in all domains of life. This modification is present in nearly all ANN-decoding tRNAs (one exception is tRNA iMet in bacteria) (2). Like other modifications found at position 37, such as m 1 G, i 6 A, or ms 2 i 6 A modifications (3,4), t 6 A is important for translation fidelity because it stabilizes the ASL structure and enhances codon-anticodon interaction (5).…”

“…The enzymatic synthesis of t 6 A has been studied in vitro and in vivo for more than 40 years (2,(6)(7)(8)(9), but the synthesis pathways were only recently elucidated, revealing both a core set of enzymes and kingdom-specific variations ( Fig. 1) (10,11).…”