RNA-specific ribonucleotidyl transferases

Abstract

RNA-specific nucleotidyl transferases (rNTrs) are a diverse family of template-independent polymerases that add ribonucleotides to the 3'-ends of RNA molecules. All rNTrs share a related active-site architecture first described for DNA polymerase beta and a catalytic mechanism conserved among DNA and RNA polymerases. The best known examples are the nuclear poly(A) polymerases involved in the 3'-end processing of eukaryotic messenger RNA precursors and the ubiquitous CCA-adding enzymes that complete the 3'-ends of tRNA molecules. In recent years, a growing number of new enzymes have been added to the list that now includes the "noncanonical" poly(A) polymerases involved in RNA quality control or in the readenylation of dormant messenger RNAs in the cytoplasm. Other members of the group are terminal uridylyl transferases adding single or multiple UMP residues in RNA-editing reactions or upon the maturation of small RNAs and poly(U) polymerases, the substrates of which are still not known. 2'-5'Oligo(A) synthetases differ from the other rNTrs by synthesizing oligonucleotides with 2'-5'-phosphodiester bonds de novo.

FIGURE 1.

Human ribonucleotidyl transferases. (Names in red) Canonical and (blue) noncanonical rNTrs. Cellular localizations (loc) are abbreviated as C (cytoplasmic), N (nuclear), M (mitochondrial), and No (nucleolar). (Uppercase letters) Experimentally demonstrated and (lowercase letters) predicted localization; (unk) unknown; (signal reg.) signal regulator. The SWISS-PROT accession code is indicated in the “access” column. PDB structure database codes (pdb) of structures from the most similar organism are indicated with lowercase letters or with uppercase letters if the structure of the human protein is available. Color code for domains: (orange) catalytic domain; (light blue) central domain; (violet) RBD; (red) nucleotide recognition motif (NRM type 1) in canonical rNTrs; (yellow) NRM type 2 in noncanonical rNTrs; (purple) NRM in CCAtrs; (cyan) zinc finger motifs; (olive) insert in U6 TUTase; (hatched bars) predicted inactive NTr motifs.

FIGURE 2.

Catalytic and central domains and RNA-binding domains (RBDs) of canonical and noncanonical rNTrs. (A) Bovine PAP pdb:1Q78 (Martin et al. 2004) with bound 3′-dATP. Color code for domains: (orange) catalytic domain (CAT); (blue) central domain (CD); and (green) RBD. Color codes are the same for B–D. The domain nomenclature used by the authors of these structures may differ. (B) 2′-5′-Oligo(A) synthetase (pdb:1PX5) (Hartmann et al. 2003) with ATP modeled from A. (C) Trypanosoma brucei RET2 (pdb:2B56) (Deng et al. 2005) with UTP. (D) Vaccinia virus PAP (pdb:2GA9) (Moure et al. 2006) with bound ATP-γ-S.

FIGURE 3.

Nucleotide base recognition motifs in human rNTrs. (Red shading) The main nucleotide-contacting residue, (blue) small, (yellow) hydrophobic, and (orange) aliphatic residues. The sequences included in the alignment correspond to the proteins in Figure 1 and are named by SWISS-PROT accession.

FIGURE 4.

Comparison of catalytic sites of canonical and noncanonical rNTrs. (A) Bovine PAP (pdb:1Q78) (Martin et al. 2004), apparent nonspecific nucleotide recognition. (Red dotted lines) Metal coordination; (green dotted lines) hydrogen bonds (light pink dotted lines indicate weak hydrogen bonds; see text); (cyan spheres) water; (gray spheres) Mg ions. Only waters relevant for substrate binding are displayed. (B) RET2 (pdb:2B56) (Deng et al. 2005) suggesting specific recognition of UTP via water molecules. The same color code applies as for A. Molecular graphics were done with PyMOL (DeLano 2002).

FIGURE 5.

The Pol β-like nucleotidyltransferase superfamily (adapted from Aravind and Koonin 1999). (Red) Protein families relevant to this review. Abbreviations in the circles are (TRF) Trf4-like poly(A) polymerases, including GLD-2 and TUTases; (OAS) 2′-5′-oligo(A) synthetases; (CCA arch) archaeal CCAtrs; (PAP euk) eukaryotic canonical PAPs; (PAP pox) poxviral PAPs; (CCA euk) eukaryotic CCAtrs; (PAP bac) bacterial PAPs; (CCA bac) bacterial CCAtrs; (CCA pla) plant CCAtrs; (CC-add) CC-adding enzymes; (A-add) A-adding enzymes; (MNT) minimal nucleotidyl transferases. Families (in black) not treated in this review are (polX) DNA polymerases of family X, including Pol β; (TdT) terminal deoxynucleotide transferase; (Str NT) streptomycin nucleotidyl transferase and (Kan NT) kanamycin nucleotidyl transferase (antibiotics resistance factors); (GlnD) protein uridylyl-transferases; (GlnE) protein adenylyl-transferases; (Sig-NT) putative signal transducing NTrs in prokaryotes; (CyA) γ-proteobacterial adenylate cyclases (Aravind and Koonin 1999). Thick connection lines indicate high (e-values < 0.01) and broken lines low statistical significance of similarity between two families (black lines are from Aravind and Koonin 1999 and red lines are from G. Martin, unpubl.). The phylogenetic relationships among the groups at the lower left is arbitrary and not statistically proven. (Pale blue background) Class I and (orange background) class II NTrs. Modified from Fig. 1 in Aravind and Koonin (1999) and reprinted with permission from Oxford University Press ©1999.

FIGURE 6.

Two structure models of class I and class II CCAtrs complexed with tRNAs and the reshaping active site of A. fulgidus CCAtr with bound tRNA 3′-end and CTP. (A) The class I A. fulgidus structure model in complex with tRNA (orange; pdb accession 1SZ1) (Xiong and Steitz 2004); (H) head domain, (N) neck, (B) body, and (T) tail. The polypeptide is colored in rainbow colors from dark blue at the N terminus via green, yellow, orange to red at the C terminus. (B) Class II CCAtr from Aquifex aeolicus with (gray) tRNA modeled instead of the cocrystallized minihelix (pdb accession 1VFG) (Tomita et al. 2004). Coloring of the polypeptide is as in D. (C) Active-site pocket with tRNA-DC74 and CTP. Two gray residues of the tRNA (at the right) followed by (pale green) D73 (the discriminator base), C74, and (orange) adjacent CTP. (Cyan sphere) A metal ion. (D) (pale green) Discriminator base D73 followed by (yellow) C74, (orange) C75, and with (red) bound ATP and (cyan sphere) a metal ion.