UTP-bound and Apo Structures of a Minimal RNA Uridylyltransferase

Abstract

Summary3′-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase β. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 Å crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 Å crystal structure of TbTUT4 with the bound 2′ deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides.

Targeted depletion of a mitochondrial nucleotidyltransferase suggests the presence of multiple enzymes that polymerize mRNA 3′ tails in Trypanosoma brucei mitochondria

“…As stated above kPAP2 also displays homology to known TUTases. However, multiple alignment of the kPAP2 amino acid sequence with those of RET1, RET2, and TUT3 reveals that kPAP2 lacks the ~100 nt insertion found in the middle domain of these TUTases (data not shown) (47). Absence of this TUTase-specific insertion is consistent with a role for kPAP2 in polyadenylation as opposed to uridylation.…”

“…The next highest match, TbTUT4, had a score of 2.7e-07. However, TbTUT4 has been recently demonstrated to be a cytoplasmic TUTase (47), and is unlikely to be involved in mitochondrial polyadenylation. No other protein with an E-value lower than e-5, which is the typical threshold for highly unique alignments, was identified by the search.…”

Structure and function of the polymerase core of TRAMP, a RNA surveillance complex

“…2C) and the terminal uridyltransferases RET2 and TUT4 from Trypanosoma brucei (16,17). Trf4p differs from these RNA polymerases most notably near the Air2p binding surface, in a longer helix H9 and in the length and conformation of the connector between helices H6 and H8 (residues 357-372 in Trf4p).…”

Determinants of substrate specificity in RNA-dependent nucleotidyl transferases

“…Fig. 3A depicts the example of the T. brucei TUT4 (TbTUT4) active site with bound UTP [28] that is very similar to the RET2 active site [29]. The triphosphate moiety in the TbTUT4 structure contributes three non-bridging phosphate oxygens to the coordination of Mg 2+ , whereas two carbonyl oxygens of Asp66 and Asp68 and an additional water complete the metal coordination shell.…”

“…The results also suggest that the penultimate U of the primer is in fact bound via hydrogen bonds to Arg126, which is a U specific interaction. This could be a control function to ensure error free poly(U) synthesis and it was demonstrated that mutation of Arg126 results in a completely inactive enzyme because of loss of RNA binding but not UTP binding [28].…”