Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases

Abstract

Terminal RNA uridylyltransferases (TUTases) catalyze templateindependent UMP addition to the 3 hydroxyl of RNA. TUTases belong to the DNA polymerase ␤ superfamily of nucleotidyltransferases that share a conserved catalytic domain bearing three metal-binding carboxylate residues. We have previously determined crystal structures of the UTP-bound and apo forms of the minimal trypanosomal TUTase, TbTUT4, which is composed solely of the N-terminal catalytic and C-terminal base-recognition domains. Here we report crystal structures of TbTUT4 with bound CTP, GTP, and ATP, demonstrating nearly perfect superposition of the triphosphate moieties with that of the UTP substrate. Consequently, at physiological nucleoside 5-triphosphate concentrations, the protein-uracil base interactions alone are not sufficient to confer UTP selectivity. To resolve this ambiguity, we determined the crystal structure of a prereaction ternary complex composed of UTP, TbTUT4, and UMP, which mimics an RNA substrate, and the postreaction complex of TbTUT4 with UpU dinucleotide. The UMP pyrimidine ring stacks against the uracil base of the bound UTP, which on its other face also stacks with an essential tyrosine. In contrast, the different orientation of the purine bases observed in cocrystals with ATP and GTP prevents this triple stacking, precluding productive binding of the RNA. The 3 hydroxyl of the bound UMP is poised for in-line nucleophilic attack while contributing to the formation of a binding site for a second catalytic metal ion. We propose a dual role for RNA substrates in TUTase-catalyzed reactions: contribution to selective incorporation of the cognate nucleoside and shaping of the catalytic metal binding site.crystal structure ͉ nucleotidyl transferase ͉ RNA editing ͉ Trypanosoma ͉ terminal RNA uridylyltransferase poly(A) polymerase T erminal RNA uridylyltransferases (TUTases) are phylogenetically widespread and functionally divergent enzymes that catalyze template-independent transfer of UMP residues to the 3Ј hydroxyl group of RNA (1). TUTases belong to the polymerase ␤ nucleotidyltransferase superfamily, which is characterized by the presence of the signature motif hG[G/S]X9-13Dh[D/E]h (X, any; h, hydrophobic amino acids) (2) and also includes poly(A) polymerases (PAPs), ATP(CTP):tRNA nucleotidyltransferases, terminal deoxy nucleotidyltransferases, protein nucleotidyltransferases, 2Ј-5Ј oligo(A) synthetases, and antibiotic resistance nucleotidyltransferases (3). TUTase activities have been reported in mammalian cells, plants, and parasitic protists from the order Kinetoplastida, Trypanosoma brucei, and Leishmania ssp. (1). Extensive uridine insertion/deletion editing in mitochondria of trypanosomatids requires 3Ј uridylylation of guide RNAs by RNA editing TUTase 1 (RET1) (4) and insertion of Us into messenger RNAs by RNA editing TUTase 2 (RET2) (5, 6). In addition to mitochondrial RNA editing TUTases, TUT3 (7) and TUT4 (8), cytoplasmic uridylyl transferases have been reported in T. brucei. Human cells apparently possess several ...

Novel TUTase associates with an editosome-like complex in mitochondria of Trypanosoma brucei

“…The replacement of the terminal uracil in 6[U] RNA ( Fig. 3) with adenosine fully restored RET2 activity to that observed on 6[A] RNA (Stagno et al 2007a). The opposite effects were observed for MEAT1: virtually no UMP incorporation into 6[A] RNA compared with 6[U] RNA.…”

Determinants of substrate specificity in RNA-dependent nucleotidyl transferases

“…These specific interactions occur in conjunction with a sequential rearrangement of the binding pocket to accommodate the growing 3′ end and the incoming nucleotide and thus ensure the correct synthesis of CCA synthesis [35,36]. Recently reported ternary structures of PAPs and TUTases with nucleotide and primer similarly suggest that specificity for nucleotide selection results from interactions of the incoming nucleotide with active site amino acids as well as with the RNA primer [31,37].…”

“…Information on nucleotide specificity is based on structural data of binary and ternary complexes. In light of recent ternary structures, specificity for the incoming nucleotide can also be contributed by the primer [31,37]. Many of the characteristics listed in Table 1 are not precisely known and necessitate further investigation.…”

“…A structure of TbTUT4 with nucleotide and UMP (an RNA mimic) or the postreaction complex with the UpU dinucleotide ( Fig. 3B) together with biochemical tests of different types of primers suggests that a U residue is preferred at the penultimate position of the RNA substrate and that the uracil base at the 3′ terminus of the RNA primer is involved in correct U addition [31]. The authors propose a dual role for the RNA substrate: first, the ultimate uracil base at the primer's 3′ end forms a stacking interaction with the uracil base of the bound nucleotide, which also stacks with an essential tyrosine and second, that the primer contributes to the formation of a productive catalytic metal binding site.…”

“…This is surprising because adenine and uracil differ with respect to size and Watson-Crick interface. Moreover, the recently reported crystal structures of TbTUT4 in complex with the nucleotides ATP, GTP and CTP in addition to UTP indicate that nucleotide binding in these enzymes is rather promiscuous and that specificity must be obtained by additional means [31]. A structure of TbTUT4 with nucleotide and UMP (an RNA mimic) or the postreaction complex with the UpU dinucleotide ( Fig.…”