Determinants of substrate specificity in RNA-dependent nucleotidyl transferases

Abstract

Poly(A) polymerases were identified almost 50 years ago as enzymes that add multiple AMP residues to the 3' ends of primer RNAs without use of a template from ATP as cosubstrate and with release of pyrophosphate. Based on sequence homology of a signature motif in the catalytic domain, poly(A) polymerases were later found to belong to a superfamily of nucleotidyl transferases acting on a very diverse array of substrates. Enzymes belonging to the superfamily can add from single nucleotides of AMP, CMP or UMP to RNA, antibiotics and proteins but also homopolymers of many hundred residues to the 3' ends of RNA molecules. The recently reported structures of several nucleotidyl transferases facilitate the study of the catalytic mechanisms of these very diverse enzymes. Numerous structures of CCA-adding enzymes have now revealed all steps in the formation of a CCA tail at the 3' end of tRNAs. In addition, structures of poly(A) polymerases and uridylyl transferases are now available as binary and ternary complexes with incoming nucleotide and RNA primer. Some of these proteins undergo significant conformational changes after substrate binding. This is proposed to be an indication for an induced fit mechanism that drives substrate selection and leads to catalysis. Insights from recent structures of ternary complexes indicate an important role for the primer molecule in selecting the incoming nucleotide.

Fig. 1

Structures of selected RNA dependent nucleotidyl transferases. (A) MNT (light gray; PDB accession 1NO5; [7]) with ATP (green) and Mg²⁺ (gray sphere) modeled from bovine PAP structure (PDB accession 1Q78). Amino acid ligands relevant for ATP binding are depicted with nitrogen in blue and oxygen in red. (B) Bovine PAP-α structure (PDB accession 1Q78; [42]) with bound ATP (red) and Mg²⁺ (gray). Color code for the domains are: orange for catalytic domain, dark blue for central domain and green for RNA binding domain. (C) Editing TUTase RET2 from T. brucei (PDB accession 2B56; [29]). Domains functionally correspond to catalytic domain (orange), central domain (dark blue) and RBD (green) of bovine PAP. (D) Class I CCAtr of Archaeoglobus fulgidus with bound tRNA (gray; PDB accession 1SZ1; [35]). The colors correspond to the head (catalytic) domain (orange), neck domain (dark blue), body (green) and tail domain (red).

Fig. 2

Simplified phylogenetic tree of NTrs relevant to this review. Class I rNTrs include the canonical eukaryotic PAPs (euk PAP) and non-canonical PAPs (TRF) and TUTases (TUT), which are related to the archaeal CCAtrs (arch CCA). Class II rNTrs include bacterial CCAtrs (bact CCA) and PAPs (bact PAP), eukaryotic CCAtrs (euk CCA), plant PAPs and CCAtrs (plant PAP CCA), and A-adding (A-add) and CC-adding (CC-add) enzymes found in some bacteria. Minimal nucleotidyl transferases (MNT) are the ancestors of the above rNTrs.

Fig. 3

Active site structures of five representative rNTrs. Amino acid ligands are shown in yellow color with nitrogen in blue and oxygen in red, except in E, where amino acids are shown in orange if belonging to the catalytic domain and blue when from the central domain. Metal ions are depicted as gray spheres and waters as blue spheres. Nucleotides are colored green for ATP, pink for CTP and cyan for UTP or UMP. Only relevant waters are depicted. Hydrogen bonds are indicated as green dotted lines and metal coordination as red dotted lines. π-stacking interactions are visualized by red double bars and stacking interactions between adenine or uracil bases and amino acids as red double arcs in panels B, D, E and F. Incoming nucleotides are labeled ATP or UTP. (A) Minimal RNA uridylyl transferase TUT4 from T. brucei (PDB accession 2IKF; [28]) with bound UTP and Mg²⁺. (B) TUT4 from T. brucei (PDB accession 2Q0F-A; [31]) with bound UTP, UMP (primer mimic) and two Mg²⁺ ions. (C) CCA-adding enzyme of A. fulgidus with bound ATP, Mn²⁺ and primer (PDB accession 1TFW; [35]). Only four nucleotides at the 3′ end of the primer tRNA are shown and labeled as C72, A73 (D; discriminator), C74 and C75. (D) Vaccinia virus PAP in complex with ATP (VP55; PDB accession 2GA9; [43]). (E) Bovine PAP-α active site with bound ATP and Mg²⁺ (PDB accession 1Q78; [42]). (F) S. cerevisiae PAP D1154A mutant bound to ATP and (A)₅ primer; only residues A-1 to A-4 of the primer are shown. Hydrogen bonds to 2′ hydroxyl groups of primer residues A-1 to A-3 are indicated (PDB accession 2Q66; [37]).

Fig. 4

Active site geometry of Pol β (PDB accession 2FMS; [46]). Metal coordinating amino acids are shown in yellow color with nitrogen in blue and oxygen in red, metal ions as gray spheres and waters as dark blue spheres. Only residues 9 and 10 of the primer are shown (P9 and P10). In this ternary complex structure the incoming nucleotide is non-hydrolyzable, which allowed the use of a natural deoxynucleoside monophosphate at the 3′ end of the primer (dTMP). The 3′ OH participates in the ligation of the metal ion. The template strand is not shown for clarity.

Fig. 5

Examples of predicted complexes of rNTrs with specificity or processivity factors and RNA substrates (black lines). (A) Vaccinia virus PAP catalytic subunit (VP55) and processivity factor VP39 in complex with RNA primer [43]. (B) Mammalian canonical PAP consisting of catalytic domain (PAP-cat) with hidden central domain and RNA binding domain (RBD) in complex with FIP1 (green) and PABPN1 (yellow) and with bound RNA primer [53, 58]. (C) Cytoplasmic PAP GLD-2 of C. elegans in complex with the RNA binding subunit GLD-3 and RNA [16]. (D) Trf4 and the RNA binding specificity factor Air1 or Air2 of S. cerevisiae and tRNA [19].