How a CCA sequence protects mature tRNAs and tRNA precursors from action of the processing enzyme RNase BN/RNase Z

Abstract

In many organisms, 3' maturation of tRNAs is catalyzed by the endoribonuclease, RNase BN/RNase Z, which cleaves after the discriminator nucleotide to generate a substrate for addition of the universal CCA sequence. However, tRNAs or tRNA precursors that already contain a CCA sequence are not cleaved, thereby avoiding a futile cycle of removal and readdition of these essential residues. We show here that the adjacent C residues of the CCA sequence and an Arg residue within a highly conserved sequence motif in the channel leading to the RNase catalytic site are both required for the protective effect of the CCA sequence. When both of these determinants are present, CCA-containing RNAs in the channel are unable to move into the catalytic site; however, substitution of either of the C residues by A or U or mutation of Arg(274) to Ala allows RNA movement and catalysis to proceed. These data define a novel mechanism for how tRNAs are protected against the promiscuous action of a processing enzyme.

Keywords: Enzyme Structure; Nucleic Acid Chemistry; Nucleic Acid Enzymology; Phosphodiesterases; Protein-Nucleic Acid Interaction; RNA Catalysis; RNA Processing; RNA-Protein Interaction; Structural Biology; Transfer RNA (tRNA).

FIGURE 1.

Action of RNase BN on CCA-containing and various CCA-less model RNA substrates. Reactions were carried out as described under “Experimental Procedures” using 5′-³²P-labeled single-stranded model RNA substrates with the sequence G₅A₁₂XA₅ (10 μm). Digestion was carried out with purified RNase BN (1.7 μm) in the presence of Mg²⁺, and portions were withdrawn at 0, 20, and 40 min. Digestion products were analyzed by 20% denaturing PAGE. The numbers at the bottom of the gel are the lane numbers, and the numbers on the side are the chain lengths of standards (25 or 18) or of a product (6).

FIGURE 2.

Structure of the RNase BN catalytic channel. The top panel shows the structure of E. coli RNase BN (Protein Data Bank entry 2CBN) in a ribbon representation with the two subunits colored pale green and pale orange. To model the path of an RNA substrate downstream of the RNase BN catalytic site (indicated by the two zinc ions coordinated at the active site shown as blue spheres), we used the structure of tRNA bound to B. subtilis RNase Z (23) (Protein Data Bank entry 4GCW). Residues 69–74 of this tRNA^Thr precursor, positioned on RNase BN by superposition of the 4GCW structure on 2CBN, are shown in a stick representation. The bottom panel shows an expanded view of the catalytic channel leading into the catalytic site. Amino acids in the channel that interact with the RNA are shown. Note that RNA residues Cys72 and Gly71, two and three nucleotides downstream of the catalytic site, are located within H-bonding distance of Arg²⁷⁴ when positioned as a single strand in a syn conformation (base rotated from a glycosyl bond torsion angle of 158.6° in the 4GCW structure to 18°).

FIGURE 3.

Members of RNase Z family contain a conserved HXSXR motif located near the catalytic site. A structure-based sequence alignment was generated by T-Coffee using sequences of RNase Z proteins from 22 different species. ESPript was used to present the sequence alignment (26). The sequences are from the Uniprot database. The sequences included are: RBN_ECOLI, E. coli RNase BN (entry no. P0A8V0); UniRef90_C4BUZ1, Enterobacteriaceae RNase BN; F7RBB1_SHIFL, Shigella flexneri RNase BN (F7RBB1); B7LM62_ESCF3, Escherichia fergusonii RNase BN (B7LM62); RBN_SALA4, Salmonella agona RNase BN (B5EZJ2); C1M711_9ENTR, Citrobacter sp. RNase BN (C1M711); D6DRB4_ENTCL, Enterobacter cloacae RNase BN (D6DRB4); RBN_KLEP3, Klebsiella pneumonia RNase BN (B5XNW7); H5UXS8_ESCHE, Escherichia hermannii RNase BN (H5UXS8); RBN_ERWT9, Erwinia tasmaniensis RNase BN (B2VHF1); RNZ_BACSU, B. subtilis RNase Z (P54548); NZ_BACAN, Bacillus anthracis RNase Z (Q81M88); NZ_BACC2, Bacillus cereus RNase Z (B71WQ5); RNZ_ENTFA, Enterococcus faecalis RNase Z (Q834G2); RNZ_LACLA, Lactococcus lactis RNase Z (Q9CHT8); RNZ_STRZJ, Streptococcus pneumonia RNase Z (C1CD41); RNZ_SYNY3, Synechocystis sp. RNase Z (Q55132); NZ_MICAN, Microcystis aeruginosa RNase Z (B0JGG3); RNZ_STAEQ, Staphylococcus epidermidis RNase Z (Q5HP47); RNZ_LACAC, Lactobacillus acidophilus RNase Z (Q5FKH3); RNZ2_MOUSE, Mus musculus ElaC2 protein (Q80Y81); and RNZ2_MOUSE, Homo sapiens ElaC2 (Q9BQ52). The stars indicate a conserved motif present in the catalytic channel. Other conserved residues are highlighted including the catalytic site residues Asp²¹² and His²⁷⁰.

FIGURE 4.

Activity of wild type and R274A mutant RNase BN on CCA-containing mature and precursor model tRNA substrates. 5′-³²P-labeled single-stranded model RNA substrates (10 μm) with the sequence G₅A₁₂CCA (A) or G₅A₁₂CCAA₅ (B) were digested with wild type or mutant RNase BN (13.6 μm) as described under “Experimental Procedures.” Portions were withdrawn at the indicated times and analyzed by denaturing PAGE. The values at the bottom of each lane indicate the percentage of the initial substrate remaining. Note that the band labeled 6 is the limit product generated by the WT enzyme.

FIGURE 5.

Action of wild type and R274A mutant RNase BN on tRNA^Phe. A, structure of tRNA^PheV is shown with the 3′-terminal CCA sequence in bold type. The 3′-terminal [³²P]C residue used in C is in parentheses. B, wild type and mutant RNase BN (1.12 μm) were used to digest full-length tRNA^Phe (∼0.05 μm). Portions were withdrawn at 0, 1.5, and 3 h. Cleavage products were analyzed by 6% denaturing PAGE, followed by Northern blotting using a 5′-probe. M, full-length tRNA. C, full-length tRNA (∼0.05 μm), labeled with [³²P]pC at its 3′-end, was treated with wild type and mutant RNase BN (1.12 μm) for 0, 1.5, and 3 h. Digestion products were analyzed by 20% denaturing PAGE. P, precursor tRNA.

FIGURE 6.

Schematic representation of the cleavage of CCA-containing and CCA-less tRNAs at the catalytic site of RNase BN. cat site is the catalytic site of RNase BN, and N represents either A or U residues replacing C residues in the CCA sequence. The arrows indicate the sites of RNA cleavage after the discriminator nucleotide (D) of tRNA. 1, Arg²⁷⁴ of wild type RNase BN interacts with adjacent C residues at the 3′-end of tRNA and prevents tRNA from moving into the catalytic site. 2 and 3, mutation of Arg²⁷⁴ to Ala (step 2) or substitution of adjacent C residues with A or U residues (step 3) allows the 3′-end of tRNA to move into the catalytic site enabling cleavage to occur after the discriminator nucleotide. Bis(p-nitrophenyl) phosphate, the phosphodiesterase (PDase) substrate, binds to the catalytic site when Arg²⁷⁴ and CCA are present (1) but is unable to bind when either one of these two determinants is absent (steps 2 and 3).

FIGURE 7.

Phosphodiesterase activity of wild type and R274A mutant RNase BN in the presence of RNA oligonucleotides. A, phosphodiesterase activity of wild type and R274A mutant RNase BN was measured as described under “Experimental Procedures” in the presence of the indicated concentrations of model RNAs. B, phosphodiesterase activity of wild type RNase BN in the presence of model RNAs with different 3′-ends, used at the indicated concentrations. Phosphodiesterase activity in the absence of RNA was set at 100. The values shown are the averages of three independent experiments.