tRNAHis guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition

Abstract

Yeast tRNA(His) guanylyltransferase, Thg1, is an essential protein that adds a single guanine to the 5' end (G(-1)) of tRNA(His). This G(-1) residue is required for aminoacylation of tRNA(His) by histidyl-tRNA synthetase, both in vitro and in vivo. The guanine nucleotide addition reaction catalyzed by Thg1 extends the polynucleotide chain in the reverse (3'-5') direction of other known polymerases, albeit by one nucleotide. Here, we show that alteration of the 3' end of the Thg1 substrate tRNA(His) unleashes an unexpected reverse polymerase activity of wild-type Thg1, resulting in the 3'-5' addition of multiple nucleotides to the tRNA, with efficiency comparable to the G(-1) addition reaction. The addition of G(-1) forms a mismatched G.A base pair at the 5' end of tRNA(His), and, with monophosphorylated tRNA substrates, it is absolutely specific for tRNA(His). By contrast, reverse polymerization forms multiple G.C or C.G base pairs, and, with preactivated tRNA species, it can initiate at positions other than -1 and is not specific for tRNA(His). Thus, wild-type Thg1 catalyzes a templated polymerization reaction acting in the reverse direction of that of canonical DNA and RNA polymerases. Surprisingly, Thg1 can also readily use dNTPs for nucleotide addition. These results suggest that 3'-5' polymerization represents either an uncharacterized role for Thg1 in RNA or DNA repair or metabolism, or it may be a remnant of an earlier catalytic strategy used in nature.

Fig. 1.

Nucleotide addition reactions catalyzed by 5′-3′ polymerases and tRNA^His guanylyltransferase (Thg1).

Fig. 2.

Thg1 exhibits 3′-5′ reverse polymerase activity with tRNA^His variants containing altered 3′ ends. (A) Schematic of Thg1 assay with 5′-³²P-labeled tRNA^His. After RNase A and phosphatase treatment, the Thg1 reaction products [G[³²P]pGpC (Gp*GpC) and longer species] are resolved from ³²P_i derived from substrate by TLC. (B) Thg1 adds guanine nucleotides to A₇₃C tRNA^His variants. Activity with 5′-³²P-labeled wild-type tRNA^His (a), tRNA^His C₇₃CCA (b), tRNA^His C₇₃CAA (c), or tRNA^His C₇₃ACA (d) variants is shown using 5-fold serial dilutions of purified Thg1 (50 μg/ml to 0.4 μg/ml); −, no Thg1 added. (C) Confirmation of identity of additional 5′ reaction products formed by Thg1. Comigration of tRNA^His C₇₃CCA G₋₁, G₋₂, and G₋₃ nucleotide addition products with [3′-³²P]Cp-labeled, calf intestinal alkaline phosphatase (CIP)-treated oligonucleotide standards (GpGp*C, GpGpGp*C, and GpGpGpGp*C; lanes c, a, and b, respectively) is shown after resolution by silica TLC.

Fig. 3.

Thg1 adds guanine nucleotides to the 5′ end of tRNA^His variants in a template-dependent reaction. (A) Thg1 reverse polymerization does not require ATP with ppp-tRNA. Primer extension analysis of Thg1 reaction products is shown with wild-type (a), C₇₃CCA (b), C₇₃CAA (c), or C₇₃ACA (d) ppp-tRNA^His species in the presence of ATP (a–d) or in the absence of ATP (e). The horizontal line denotes the expected primer extension stop with unreacted transcript (position +1), and additional nucleotide incorporations are indicated by (−1), (−2), and (−3). The primer position is indicated by an arrow on the tRNA diagram; G₋₁, G₋₂, and G₋₃ nucleotides incorporated by Thg1 with the tRNA^His C₇₃CCA variant are indicated in parentheses. (B) Thg1 reverse polymerization does not depend on the source of purified Thg1 protein. Shown is the primer extension of reaction products from the addition of G₋₁ (with wild-type tRNA^His) and reverse polymerization (with tRNA^His C₇₃CCA) using yeast Thg1 purified from either Saccharomyces cerevisiae (Sc) or Escherichia coli (Ec). (C) Thg1 adds guanosine residues according to the number of C residues at the 3′ end of the tRNA species. Thg1 reaction products with tRNA^His C₇₃CCCCA were analyzed by primer extension, demonstrating the addition of up to five nucleotides to the 5′ end of the tRNA.

Fig. 4.

Nucleotide dependence of Thg1 reverse polymerization compared with the addition of G₋₁. (A) Nucleotide dependence of Thg1 activity with ppp-tRNA^His species. Thg1 reaction products formed in the presence of individual NTPs and ppp-tRNA^His substrates (with 3′ end sequence indicated below each panel) were analyzed by primer extension. Lanes G, A, U, and C show each NTP added as indicated; −, no nucleotide; NP, no Thg1. (B) Thg1 is specific for the addition of G at position −1 in the presence of all four NTPs. Thg1 reaction products with 5′-³²P-labeled wild-type tRNA^His are shown in the presence of GTP and ATP (a) or a mixture of all four NTPs (GTP, ATP, UTP, and CTP) (b–d), each at a final concentration of 1 mM (b), 0.4 mM (c), or 0.1 mM (d); −, no added nucleotide; NP, no Thg1. (Left) RNase A and CIP treatment of reaction products, resolved by standard silica TLC. (Right) RNase T2 digestion of reaction products, to release 3′-labeled Np resulting from the addition of nucleotide at position −1, resolved by polyethyleneimine (PEI)-cellulose TLC in 0.5 M sodium formate (pH 3.5). Gp, Ap, Up, and Cp, migration of 3′-phosphorylated standards as visualized by fluorescence quenching.

Fig. 5.

Thg1 reverse polymerase activity is not specific for tRNA^His and does not need to begin at the −1 position. (A) tRNA^Phe C₇₃CCA is a substrate for Thg1 reverse polymerization. Thg1 catalyzes the addition of G₋₁ to wild-type ppp-tRNA^Phe and addition of multiple G residues to ppp-tRNA^Phe C₇₃CCA, as analyzed by primer extension. (B) Reverse polymerization can initiate at position −1. Thg1 catalyzes addition of G₋₂ and G₋₃ to a tRNA^His 76-mer transcript that already contains G₋₁ when this G₋₁ residue is base-paired with C₇₃, but not when it is opposite A₇₃, as analyzed by primer extension. (C) Reverse polymerization can initiate at position +2. Thg1 catalyzes the addition of G₊₁ and G₋₁ to a tRNA^His variant that begins at the +2 position, yielding the same G₋₁-containing reaction product as a control RNA species that begins with G₊₁, as analyzed by primer extension. (D) Thg1 does not add additional residues to p-tRNA^His species that do not begin with G₊₁. 5′-³²P-labeled wild-type or variant tRNA^His and tRNA^Phe species, as indicated below each panel, were assayed in the presence of GTP and ATP. GpGpC is the reaction product observed with wild-type tRNA^His; GpGpGpC is the tetramer RNase A reaction product expected for G₊₂ control RNA, which has an altered G₂·C₇₁ base pair.

Fig. 6.

Thg1 adds deoxynucleotides and ribonucleotides to the 5′ end of tRNA^His. (A) Comparison of Thg1 activity in the presence of GTP or dGTP. Thg1 was assayed with 5′-³²P-labeled wild-type or C₇₃CCA tRNA^His and the standard RNase A/CIP assay in the presence of either GTP or dGTP as indicated. (B) Verification of deoxyguanosine nucleotide incorporation into tRNA. (a) Samples from A were resolved by TLC on PEI-cellulose in 0.5 M sodium formate (pH 3.5), where dG-containing reaction products migrate differently from the corresponding ribonucleotide-containing products. (b) Samples from A were treated with RNase T1 and CIP and resolved in the same system.