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

Abstract

The Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex recognizes aberrant RNAs in Saccharomyces cerevisiae and targets them for degradation. A TRAMP subcomplex consisting of a noncanonical poly(A) RNA polymerase in the Pol ss superfamily of nucleotidyl transferases, Trf4p, and a zinc knuckle protein, Air2p, mediates initial substrate recognition. Trf4p and related eukaryotic poly(A) and poly(U) polymerases differ from other characterized enzymes in the Pol ss superfamily both in sequence and in the lack of recognizable nucleic acid binding motifs. Here we report, at 2.7-A resolution, the structure of Trf4p in complex with a fragment of Air2p comprising two zinc knuckle motifs. Trf4p consists of a catalytic and central domain similar in fold to those of other noncanonical Pol beta RNA polymerases, and the two zinc knuckle motifs of Air2p interact with the Trf4p central domain. The interaction surface on Trf4p is highly conserved across eukaryotes, providing evidence that the Trf4p/Air2p complex is conserved in higher eukaryotes as well as in yeast and that the TRAMP complex may also function in RNA surveillance in higher eukaryotes. We show that Air2p, and in particular sequences encompassing a zinc knuckle motif near its N terminus, modulate Trf4p activity, and we present data supporting a role for this zinc knuckle in RNA binding. Finally, we show that the RNA 3' end plays a role in substrate recognition.

Fig. 1.

The Air2p zinc knuckles modulate the activity of Trf4p. (A) Representative gels show a time course for polyadenylation, where 2 pmol Trf4p/Air2p complex is reacted with 40 fmol of wild-type (lanes 1–6, 13–18) or mutant (lanes 7–12, 19–24) . Time courses are for Trf4p/Air2p_ZK1-5 (ZK1-5, lanes 1–12) and Trf4p/Air2p_ZK4-5 (ZK4-5, lanes 13–24). (B) Plot showing the fraction of adenylated at given time points. The data are from three separate experiments. SEM is indicated. (C) Mutant was reacted with Trf4p/Air2p_ZK1-5, Trf4p/Air2p_ZK4-5, or Trf4p/Air2p_ZK1-5 but with the three most N-terminal knuckles individually replaced by hexaserine linkers (ZK1mut, ZK2mut, ZK3mut). (D) Quantitation of three experiments as in C. SEM indicated. (E) A₅ oligonucleotide is polyadenylated comparably by all Trf4p/Air2p complexes used in C and D.

Fig. 2.

Structure of a Trf4p/Air2p subcomplex at 2.7-Å resolution. (A) Schematic of Trf4p and Air2p, indicating regions included in the crystallization construct. Air2p zinc knuckles are boxed. (B) Ribbons diagram with the catalytic and central domains of Trf4p in cyan and blue, respectively. Orange dots indicate positions of the aspartate residues in the catalytic triad. The fourth and fifth zinc knuckles of Air2p and adjacent linker regions (ZK4, ZK5) are yellow and green, respectively, and zincs are red. (C) Superposition of the Trf4p/Air2p subcomplex (blue/yellow and green) with another noncanonical RNA polymerase, CCA-adding enzyme from A. fulgidus (PDB ID 2DRA, cyan). The catalytic and central domains were superimposed separately because their relative orientation differs in the two structures.

Fig. 3.

The Air2p-interaction surface of Trf4p is conserved in eukaryotes. (A) Space-filling model of Trf4p with surface residues within 4.5 Å of Air2p zinc knuckles colored lime or green. Air2p zinc knuckles are shown as worms (fourth, yellow; fifth, green), and Air2p side chains that interact with Trf4p are labeled. (B) As in A, but residues in Trf4p that are within 4.5 Å of Air2p are labeled and Air2p is not illustrated. (C) Residues that are identical or similar in Trf4p and six of seven other sequence-related polymerases are labeled and colored blue and cyan, respectively. We compared the sequence of Trf4p with sequences from S. cerevisiae, S. pombe, humans, Gallus gallus, Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans. An alignment is in Fig. S3. Trf4p is oriented as in A and B. (D and E) Surface conservation as in C, but Trf4p is differently oriented. The conserved aspartate residues in the polymerase catalytic triad are red. Panels are enlarged in Fig. S4.

Fig. 4.

RNA substrates of Trf4p/Air2p. (A) Polyadenylation of 5′ end-labeled oligomers A₁₀, C₁₀, G₁₀, or U₁₀ (30 fmol) by ∼2 pmol of Trf4p/Air2p_ZK4-5 (lanes 2, 5, 8, and 11), Trf4p/Air2p_ZK1-5 (lanes 3, 6, 9, and 12), or no protein (lanes 1, 4, 7, and 10). (B) Quantitation from three experiments in A, showing fraction of adenylated RNA. SEM is indicated. Trf4p/Air2p polyadenylates C₁₀ less extensively than A₁₀, G₁₀, or U₁₀. (C) Polyadenylation assay, where Trf4p/Air2p (2 pmol) was mixed with RNA duplexes (30 fmol) containing no overhang (lanes 1–3) or 3′- A-overhangs of length indicated (lanes 4–18). Longer extensions were added only when the 3′ overhang was three nucleotides or longer. Reactions included Trf4p/Air2p_ZK4-5 (lanes 2, 5, 8, 11, 14, and 17), Trf4p/Air2p_ZK1-5 (lanes 3, 6, 9, 12, 15, and 18), or no protein (lanes 1, 4, 7, 10, 13, and 16).