Identification of the 3-amino-3-carboxypropyl (acp) transferase enzyme responsible for acp3U formation at position 47 in Escherichia coli tRNAs

Abstract

tRNAs from all domains of life contain modified nucleotides. However, even for the experimentally most thoroughly characterized model organism Escherichia coli not all tRNA modification enzymes are known. In particular, no enzyme has been found yet for introducing the acp3U modification at position 47 in the variable loop of eight E. coli tRNAs. Here we identify the so far functionally uncharacterized YfiP protein as the SAM-dependent 3-amino-3-carboxypropyl transferase catalyzing this modification and thereby extend the list of known tRNA modification enzymes in E. coli. Similar to the Tsr3 enzymes that introduce acp modifications at U or m1Ψ nucleotides in rRNAs this protein contains a DTW domain suggesting that acp transfer reactions to RNA nucleotides are a general function of DTW domain containing proteins. The introduction of the acp3U-47 modification in E. coli tRNAs is promoted by the presence of the m7G-46 modification as well as by growth in rich medium. However, a deletion of the enzymes responsible for the modifications at position 46 and 47 in the variable loop of E. coli tRNAs did not lead to a clearly discernible phenotype suggesting that these two modifications play only a minor role in ensuring the proper function of tRNAs in E. coli.

Identification of the enzymes responsible for m2,2G and acp3U formation on cytosolic tRNA from insects and plants

“…Our ability to detect the m 2,2 G 26 modification in wild-type cells from a background of trm1Δ cells indicates that this technique is sensitive enough to detect cells with a tRNA modification from a pool of cells lacking the modifications, which could be useful in the development of a screen to identify genes required for tRNA modifications. This sensitivity also further suggests that in many instances fluorescence could be a viable alternative to traditional primer extension [78,86], which uses 32 P. 32 P has exquisite sensitivity, and is critical for the study of tRNA modifications. However, the use of radioactivity has several drawbacks, which include expense, safety concerns requiring extensive additional training of personnel, and a relatively short window in which the radioactivity can be used, due to the short half-life of 32 P [86].…”

“…DTWD2 belongs to the TDD superfamily of proteins, and is localized to the nucleus and the cytoplasm [78,106,107]. This protein superfamily contains E. coli TuaA/TapT, as well as Tsr3, which is required to add the acp group to the 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine modification (m 1 acp 3 C) found at residue U 1191 on the yeast 18s rRNA and residue U 1248 on human 18s rRNA.…”

“…This protein superfamily contains E. coli TuaA/TapT, as well as Tsr3, which is required to add the acp group to the 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine modification (m 1 acp 3 C) found at residue U 1191 on the yeast 18s rRNA and residue U 1248 on human 18s rRNA. [78,107]. TDD superfamily proteins contain a DTW domain with an E/DXS/TW motif [78,106] (Fig 5A).…”

“…In previously studied TDD proteins, the Asp/Glu and Trp residues of the motif are found in the active site and are required for catalysis. Indeed, Asp137 of this motif in E. coli TuaA/TapT is required for activity, and is likely the catalytic base, while Trp140 is required for activity and S-adenosyl methionine (SAM) cofactor binding [78,107]. To determine if the corresponding Asp and Trp residues of the motif are required for the activity of a eukaryotic DTWD2 protein (Fig 5A), we generated D133A and W136A variants of A. thaliana DTW2a and tested their modification activity.…”

“…In bacteria, this modification increases thermal stability [76]. The E. coli protein YfiP (now named TuaA or TapT) was recently identified as the enzyme that forms acp 3 U 47 on bacterial tRNA [76,78], and lack of this modification results in a motility defect and genome instability in cells [76].…”