Abstract
All nucleotide polymerases and transferases catalyze nucleotide addition in a 5′ to 3′ direction. In contrast, tRNAHis guanylyltransferase (Thg1) enzymes catalyze the unusual reverse addition (3′ to 5′) of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3′–5′ addition activity to add G−1 to the 5′-end of tRNAHis, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs) are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5′-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNAHis. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNAHis maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium Bacillus thuringiensis (BtTLP). The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3′–5′ reaction with 5′-monophosphorylated tRNA necessitates first an activation step, generating a 5′-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5′-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.
Highlights
The tRNAHis guanylyltransferase (Thg1) family comprises enzymes from all three domains of life, all of which catalyze reverse addition (39–59) of nucleotides to polynucleotide substrates [1]
By analogy to hTHG1, we propose that this second bound NTP represents the position of the incoming NTP to be added to the polynucleotide chain, which is supported experimentally by dramatic decreases in the kinetic efficiency of the nucleotidyl transfer step after alteration to alanine of several of the analogous positively charged residues in yeast Thg1 (R27, K96 and R133, which correspond to R28, K99 and R131 in BtTLP) [3]
Of the three yeast Thg1 residues K44, S76, and N161 shown to be important for the initial activation step of the reaction catalyzed by Saccharomyces cerevisiae Thg1 (ScThg1) (Figure S1), we have provided new structural data to rationalize a direct role for K44 in catalysis and reaffirmed the important role of S76 based on the conserved nature of the interactions between the serine residue and N7 of the activating NTP in both ATP- and GTP-bound structures obtained here
Summary
The tRNAHis guanylyltransferase (Thg1) family comprises enzymes from all three domains of life, all of which catalyze reverse addition (39–59) of nucleotides to polynucleotide substrates [1]. Many bacterial and archaeal species that contain a Thg1-like protein (TLP) do not require post-transcriptional addition of the G21 residue [8]. In these organisms, G21 is instead encoded by the tRNAHis gene, incorporated into tRNAHis during transcription and retained in the tRNA after RNase P processing, obviating the need for TLPs in tRNAHis maturation [9]. TLP enzymes differ from their eukaryotic counterparts because of their inability to form non-Watson-Crick base pairs during the 39–59 addition reaction, whereas the ability to incorporate a nontemplated G21 opposite a universally conserved A73 discriminator nucleotide in tRNAHis is a hallmark of eukaryotic Thg1-type enzyme activity. TLPs prefer to catalyze Watson-Crick template-dependent 39–59 reverse polymerization, suggesting that alternative roles for Thg family enzymes may exist that would take advantage of the ability to catalyze this unusual polymerase reaction, which occurs in the opposite direction to all other known DNA/RNA polymerases [10,11]
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