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. 2011 Mar;39(5):1833-42.
doi: 10.1093/nar/gkq976. Epub 2010 Nov 3.

tRNA 5'-end repair activities of tRNAHis guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea

Bhalchandra S Rao  1 , Emily L Maris, Jane E Jackman
Affiliations

tRNA 5'-end repair activities of tRNAHis guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea

Bhalchandra S Rao et al. Nucleic Acids Res. 2011 Mar.

Abstract

The tRNA(His) guanylyltransferase (Thg1) family comprises a set of unique 3'-5' nucleotide addition enzymes found ubiquitously in Eukaryotes, where they function in the critical G(-1) addition reaction required for tRNA(His) maturation. However, in most Bacteria and Archaea, G(-1) is genomically encoded; thus post-transcriptional addition of G(-1) to tRNA(His) is not necessarily required. The presence of highly conserved Thg1-like proteins (TLPs) in more than 40 bacteria and archaea therefore suggests unappreciated roles for TLP-catalyzed 3'-5' nucleotide addition. Here, we report that TLPs from Bacillus thuringiensis (BtTLP) and Methanosarcina acetivorans (MaTLP) display biochemical properties consistent with a prominent role in tRNA 5'-end repair. Unlike yeast Thg1, BtTLP strongly prefers addition of missing N(+1) nucleotides to 5'-truncated tRNAs over analogous additions to full-length tRNA (k(cat)/K(M) enhanced 5-160-fold). Moreover, unlike for -1 addition, BtTLP-catalyzed additions to truncated tRNAs are not biased toward addition of G, and occur with tRNAs other than tRNA(His). Based on these distinct biochemical properties, we propose that rather than functioning solely in tRNA(His) maturation, bacterial and archaeal TLPs are well-suited to participate in tRNA quality control pathways. These data support more widespread roles for 3'-5' nucleotide addition reactions in biology than previously expected.

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Figures

Figure 1.
Figure 1.
BtTLP catalyzes templated, but not non-templated addition of G-1 to tRNAHis. (A) Schematic of p*tRNAHis G−1 addition assay (24); products expected from RNase A/CIP treatment are indicated below each tRNA. The site of RNase A cleavage is indicated on each tRNA. (B) G−1 addition to A73- or C73-containing 5′-32P-tRNAHis substrates was tested using the phosphatase protection assay with serial dilutions of enzymes, as indicated, in the presence of 0.1 mM ATP and 1.0 mM GTP.
Figure 2.
Figure 2.
BtTLP catalyzes template-dependent 3′–5′ nucleotide additions to N73-tRNAHis variants. Assays for N-1 nucleotide additions contained tRNAHis variants with each of the four possible N73 discriminator nucleotides, as indicated; the tRNA diagram shows the expected positions of RNase A or RNase T1 cleavage to yield the various labeled oligonucleotide products, as indicated to the right of the figure. Reactions contained 5′-32P-labeled tRNA, 1 mM NTP (either G, A, U or C as indicated) and 0.1 mM ATP (unless ATP was already present in the assay) for activation of the 5′-monophosphorylated tRNA, and were initiated by addition of 1 µl (∼15 µg) yeast Thg1 or BtTLP. Gpp*GpC, formed by BtTLP with A73 or C73-tRNAHis, and G-2pGp*GpC, formed by yeast Thg1 with C73-tRNAHis are not resolved from each other using this TLC solvent system, but have each been verified by further digestion. Lanes dash: buffer control reactions for each N73-tRNAHis variant.
Figure 3.
Figure 3.
GTP does not compete effectively with Watson–Crick base pair forming nucleotides for N−1 addition catalyzed by BtTLP. GTP competition assays were conducted using 5′-32P labeled A73-tRNAHis or G73-tRNAHis substrates in the presence of equimolar amounts (1 mM each) of GTP and the correct Watson–Crick base pairing NTP (either UTP or CTP, as indicated). ATP (0.1 mM) was present in all reactions for 5′-monophosphate activation. Reactions were initiated with 1 µl enzyme and digested as indicated, to separately visualize purine and pyrimidine nucleotide addition products derived from the same assay, so that the ratio of templated addition to non-templated addition could be calculated for each enzyme/substrate combination.
Figure 4.
Figure 4.
BtTLP catalyzes robust G+1 addition to 5′-truncated C72-tRNAHisΔG+1. G+1 addition to 5′-32P labeled-C72tRNAHisΔG+1 was performed as described for G−1 addition assay above, using serial dilutions of yeast Thg1 (yThg1), BtTLP or MaTLP, as indicated, in the presence of 0.1 mM ATP and 1.0 mM GTP. The identity of the G+1p*GpC product was verified by migration with standards and RNase T2 digestion to release 3′-GMP (data not shown). The lower migrating products indicated by the bracket cannot be unequivocally identified due to the remote position of the labeled phosphate from the added nucleotide, but further digestions and comparison to known standards suggests that these are a mixture of further activation and addition products following G+1 addition.
Figure 5.
Figure 5.
BtTLP adds all four possible templated N+1 nucleotides to 5′-truncated tRNAHis variants (N72-tRNAHisΔN+1). (A) N+1 addition assays were performed using the same assay described in Figure 2, but with 5′-32P labeled tRNAHis variants missing a +1 nt and containing each of the four possible N72 nucleotides to serve as the template for +1 nt addition (see tRNA diagram). The single N+1 addition products produced by the relevant nuclease treatment are indicated by arrows. The full-length tRNAs generated following N+1 addition are each substrates for further activation and/or N−1 addition reactions; products of these reactions are indicated by asterisks to the right of the image, but these products are not further identified since the remote position of the labeled phosphate (between N+1 and G+2 nucleotides) does not readily permit identification by RNase T2 digestion. (B) RNase T2 digestion of reactions from (A) confirms each of the four N+1 nucleotides added to 5′-truncated tRNAHisΔN+1 substrates. RNase T2 products were resolved by PEI-cellulose TLC in 0.5 M formate, pH 3.5; positions of each 3′-32P labeled mononucleotide products (Cp*, Up*, Ap* and Gp*) were identified based on the migration of cold NMP standards. 5′-activated N+1 addition products generated from G+1 and A+1 addition reactions are indicated by NppGp* and NppAp*, respectively.
Figure 6.
Figure 6.
BtTLP catalyzes N+1 nucleotide addition to 5′-truncated tRNAHis with enhanced catalytic efficiency over N-1 addition reactions. kcat/KM values are shown for BtTLP-catalyzed addition of each of the four possible Watson–Crick templated N−1 (solid bars) or N+1 (hatched bars) nucleotides to full-length tRNAHis or 5′-truncated tRNAHisΔN+1 substrates, respectively. For each of the four nucleotides (G, C, A or U, as indicated below the figure), kinetic parameters were measured using a tRNA substrate with the appropriate N73 or N72 residue to allow Watson–Crick base paired 3′–5′ addition, as in Table 1 and Supplementary Table S1. For comparison, the kcat/KM value measured previously for yeast Thg1-catalyzed G−1 addition to C73-tRNAHis is also shown (9).
Figure 7.
Figure 7.
BtTLP catalyzes robust repair of 5′-truncated tRNAPhe substrates. The phosphatase protection assay for G+1 addition was conducted using 5′-32P-labeled C72-tRNAPheΔG+1 (see tRNA diagram) with serial dilutions of BtTLP, MaTLP or yeast Thg1 (yThg1). All reactions contained 1.0 mM GTP and 0.1 mM ATP for activation. The migration of the phosphatase-protected species is consistent with the predicted 6-nt reaction product (see diagram), also confirmed by the addition of a single nucleotide to the 5′-truncated tRNAPheΔG+1 substrate observed using primer extension (see Supplementary Figure S4).
Figure 8.
Figure 8.
Expression of BtTLP in yeast complements the growth defect of the yeast thg1Δ strain. Plasmid shuffle assays were performed with a yeast thg1Δ strain (9) transformed with CEN LEU2 plasmids containing either BtTLP [BtTLP] or yeast Thg1 [yTHG1], or no Thg1 [V1]. The top three panels also contained a second CEN HIS3 plasmid encoding [A73- tRNAHis], [C73-tRNAHis] or no tRNA [V2], as indicated. Positive transformants were grown overnight in selective media, diluted to OD600 = 1 and used to make 10-fold serial dilutions; 2 µl of each dilution was spotted to media (as indicated) and images were taken after 3–4 days of growth at 30°C.
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